EP3732184A1 - D-glycero-b-d-heptose 1-phosphate (hmp) conjugates and use for targeted immune modulation - Google Patents

D-glycero-b-d-heptose 1-phosphate (hmp) conjugates and use for targeted immune modulation

Info

Publication number
EP3732184A1
EP3732184A1 EP18896861.4A EP18896861A EP3732184A1 EP 3732184 A1 EP3732184 A1 EP 3732184A1 EP 18896861 A EP18896861 A EP 18896861A EP 3732184 A1 EP3732184 A1 EP 3732184A1
Authority
EP
European Patent Office
Prior art keywords
compound
alkyl
group
hiv
subject
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP18896861.4A
Other languages
German (de)
French (fr)
Other versions
EP3732184A4 (en
Inventor
Janelle SAUVAGEAU
Andrew Cox
Xinyi GUO
Scott Gray-Owen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
National Research Council of Canada
University of Toronto
Original Assignee
National Research Council of Canada
University of Toronto
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by National Research Council of Canada, University of Toronto filed Critical National Research Council of Canada
Publication of EP3732184A1 publication Critical patent/EP3732184A1/en
Publication of EP3732184A4 publication Critical patent/EP3732184A4/en
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H11/00—Compounds containing saccharide radicals esterified by inorganic acids; Metal salts thereof
    • C07H11/04—Phosphates; Phosphites; Polyphosphates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A61K39/005—Trypanosoma antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A61K39/008—Leishmania antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A61K39/015—Hemosporidia antigens, e.g. Plasmodium antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/0208—Specific bacteria not otherwise provided for
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/0225—Spirochetes, e.g. Treponema, Leptospira, Borrelia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/0233—Rickettsiales, e.g. Anaplasma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0258—Escherichia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0275—Salmonella
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0283—Shigella
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0291—Yersinia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/08—Clostridium, e.g. Clostridium tetani
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/085—Staphylococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/095—Neisseria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/099—Bordetella
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/118—Chlamydiaceae, e.g. Chlamydia trachomatis or Chlamydia psittaci
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/125—Picornaviridae, e.g. calicivirus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/15—Reoviridae, e.g. calf diarrhea virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/155—Paramyxoviridae, e.g. parainfluenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/205—Rhabdoviridae, e.g. rabies virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/23—Parvoviridae, e.g. feline panleukopenia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/235—Adenoviridae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/02—Phosphorylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates generally to phosphorylated heptose compounds. More specifically, the present invention relates to use of heptopyranose phosphate and conjugates thereof in modulating, or targeting the modulation of, an immune response in a subject, to improve the specificity and effect of HMP and enhance protection.
  • the immune system provides protection against infectious agents, including bacteria, viruses, fungi, and parasites.
  • infectious agents including bacteria, viruses, fungi, and parasites.
  • a substantial number of medical conditions are associated with a compromised immune system and its associated increase in susceptibility to infectious agents.
  • patients undergoing surgery, radiation or chemotherapy, and those suffering from autoimmune diseases and diseases interfering with a normal metabolic immune response, such as HIV (AIDS) are all at a heightened risk of developing pathological conditions resulting from infection.
  • pharmaceuticals - antibiotics such as ampicillin, tetracycline and quinolones
  • resistance of the infectious agent to these pharmaceuticals is an increasingly significant concern.
  • PAMPs also known as microbial-associated molecular patterns, or MAMPs
  • MAMPs microbial-associated molecular patterns
  • PAMPs lipopolysaccharide
  • CpG motifs within bacterial DNA
  • flagellin This limits the opportunity to investigate the immunomodulatory properties of these molecules.
  • PAMPs are difficult to synthesise completely or isolate at required and reproducible levels of purity, and thus precludes an opportunity to specifically address how these PAMPs interact with the immune system to exploit this relationship, when a pure, fully characterised supply of the PAMPs is unavailable.
  • the inventors have identified phosphorylated heptose compounds (such as HMP) as PAMP-like molecules useful in modulating an immune response in a subject.
  • phosphorylated heptose compounds such as HMP
  • an HMP-linker corresponding to formula (I):
  • group « R » is a linkage moiety adapted (or suitable) for conjugating with a functional molecule to form a conjugate
  • Y is H or an appropriate atom for forming a salt thereof.
  • the conjugate is adapted (or capable) of modulating an immune response in a subject. More particularly, the conjugate is adapted (or capable) of targeting a modulation of an immune response in a subject.
  • the present invention provides an immunomodulatory compound comprising a molecule conjugated to a D-glycero-D-manno-heptopyranose I b-phosphate (heptopyranose-1-monophosphate; HMP) moiety.
  • the molecule is a functional molecule, such as, for example, a drug, a cell surface antigen, a biological response modifier (BRM), a cell differentiation antigen specific binding partner or a marker.
  • the functional molecule is conjugated at position-7 of HMP or at position-2 of HMP, most particularly at position-7.
  • the invention provides a method to prepare an immunomodulator conjugate comprising the step of: conjugating a functional molecule with a compound of formula (I)
  • the immunomodulator conjugate has immunomodulatory and/or targeting activity.
  • the invention provides a conjugate compound of formula (II):
  • X is a functional molecule and Y is H or an appropriate atom for forming a salt thereof, for use in targeting and/or modulating an immune response in a subject.
  • the use for modulating an immune response in a subject may comprise: a) improving the delivery of this HMP moiety into the cell. This could be achieved by linking the HMP moiety to a lipid or fatty acid or other molecule that would improve the permeability of HMP across cell membranes; b) treating an HIV infection by stimulating HIV latently-infected cells to start producing virus, and killing the produced virus; whereby the HMP-anti-HIV conjugate eradicates the latent viral pool and treats the HIV infection; or c)
  • the invention provides a use of an effective amount of a compound of formula (II) as defined herein, for targeting and/or modulating an immune response in a subject.
  • the invention provides a use of a conjugate-compound of formula (II) for the manufacture of a medicament for targeting and/or modulating an immune response in a subject.
  • the invention provides a method for modulating an immune response in a subject, comprising administering an effective amount of the conjugate-compound of formula (II) as defined herein.
  • the invention provides a method for improving the delivery of this HMP moiety into the cell. This could be achieved by linking the HMP moiety to a lipid or fatty acid or other molecule that would improve the permeability of HMP across cell membranes
  • a method for the treatment of HIV comprising the steps of: administering an HMP-derivative conjugated to an anti-HIV molecule, wherein the HMP moiety stimulates HIV latently-infected cells to start producing virus, and wherein the anti-HIV molecule kills the produced virus; whereby the HMP-anti-HIV conjugate eradicates the latent viral pool and treats the HIV infection.
  • a method for treating latent HIV infection comprising the steps of: administering an HMP-derivative conjugated to an HIV-reservoir cell-specific targeting molecule to a subject, and treating said subject with an anti-HIV drug, wherein the HMP moiety stimulates HIV latently-infected cells to produce virus, and the anti-HIV drug kills the produced virus; whereby the method eradicates the latent HIV viral pool.
  • the invention provides a method for synthesizing a compound of formula (II) comprising the steps of Scheme 1 ( Figure 1A): Scheme 1.
  • Figure 1 B Reaction scheme 2 for the synthesis of particular embodiments of derivative linkers of HMR-b.
  • Figures 2A-B A) 1 H NMR spectra of 7-0-(Aminoethyl)-D-Glycero ⁇ -D-manno- heptopyranosyl phosphate (JS10); B) HSQC 1 H- 13 C, NMR spectra of compound JS10.
  • FIGS 3A-B A) 1 H NMR spectra of 7-0-(Aminoethyl)-D-Glycero-D-manno- heptopyranose (JS1 1 a). B) 13 C NMR spectra of 7-0-(Aminoethyl)-D-Glycero-D-manno- heptopyranose (JS1 1 a).
  • FIGS 4A-B A) 1 H NMR spectra of D-glycero-D-manno heptose (JS12a). B) 13 C NMR spectra of D-glycero-D-manno heptose (JS12a).
  • FIG. 5A-C A) 1 H NMR spectra of 7-0-(Aminoethyl-pH-Rhodo)-D-Glycero- b-D-manno-heptopyranosyl phosphate (JS13a). B) LMRS spectra of 7-0-(Aminoethyl-pH- Rhodo)-D-Glycero ⁇ -D-manno-heptopyranosyl phosphate (JS13a). C) HPLC chromatogram of 7-0-(Aminoethyl-pH-Rhodo)-D-Glycero ⁇ -D-manno-heptopyranosyl phosphate (JS13a).
  • FIGS 6A-B A) 1 H NMR spectra of 7-0-([Biotin]amidoethyl)-D-Glycero ⁇ -D- manno-heptopyranosyl phosphate (JS14a). B) 13 C NMR spectra of 7-0- ([Biotin]amidoethyl)-D-Glycero-b-D-manno-heptopyranosyl phosphate (JS14a).
  • FIGS 9A-B Inflammatory response of human colonic epithelial cells (HCT 1 16) exposed to chemically synthesized analogues.
  • Wild type (WT) or TIFA knockout (TIFA KO) cells were treated with compounds in the presence of digitonin for 20 min, treatment was removed, cells were washed and then cultured in normal medium without the synthetic compounds for 6 hr before IL-8 levels in culture supernatants were measured by ELISA.
  • A Cells were treated with water or 10 pg/mL of the indicated compound. Data are means ⁇ standard error mean of technical triplicates.
  • Nodi agonist Cl2-iE- DAP (20pg/ml, 39.8mM); JS-12: D-glycero-D-manno-heptose; JS-2: D-glycero-D-manno-heptose 7- phosphate; JS-7:D-glycero- -D-manno-heptose-phosphate; JS-8: d-glycero-a-d-manno-heptose- phosphate; JS-9: b-d-mannose phosphate; JS-6:d-glycero- -d-manno heptose 1 ,7-biphosphate; JS-5: d-glycero-a-d-manno heptose 1 ,7-biphosphate.
  • FIGS 10A-B Effect of chemically synthesised analogues on human embryonic kidney cells (HEK 293T) encoding an NF-KB-driven luciferase reporter gene.
  • NF-kB luciferase activity was measured in wild type (WT) or TIFA knockout (TIFA KO) cells treated with compounds in the presence of digitonin for 20 min, washed, and then cultured in normal medium without the synthetic compounds for 6 hrs after treatment, before luciferase activity was measured.
  • WT wild type
  • TIFA KO TIFA knockout
  • JS-12 D-glycero-D-manno-heptose
  • JS-2 D-glycero-D-manno-heptose 7-phosphate
  • JS-7 D-glycero- -D-manno-heptose-phosphate
  • JS-8 D- glycero-a-D-manno-heptose-phosphate
  • JS-9 b-D-mannose phosphate
  • JS-6 D-glycero-3-D- manno heptose 1 ,7-biphosphate
  • JS-5 D-glycero-a-D-manno heptose 1 ,7-biphosphate.
  • FIG. 11 Stimulation of human colonic epithelial cells by compounds / products according to the invention.
  • Human colonic epithelial cells HCT 1 16 that were either wild type (WT) or deficient in TIFA protein expression (knockout, KO) were stimulated for 20 minutes in permeabilization buffer (5 pg/mL digitonin) in the presence of water or 10 pg/mL, 30 pg/mL, or 50 pg/mL of indicated synthetic compounds according to the invention. Treatment was removed, cells were washed, and cells were incubated for 6 hours in complete media before IL-8 levels in culture supernatants was measured by ELISA. The results are the mean ⁇ standard error of the mean of three technical replicates.
  • JS-10 7-0-(Aminoethyl)-D-glycero-3-D-manno-heptopyranose phosphate
  • JS-11 7-0- (Aminoethyl)-D-glycero-D-manno-heptopyranose
  • JS-12 D-glycero-D-manno-heptose
  • Js-13 pH- Rhodo-7-0-(aminoethyl)-D-glycero-3-D-manno-heptopyranose phosphate.
  • Figure 12 Effects of compounds / products according to the invention on HEK 293T cells encoding an NF-KB-driven luciferase reporter gene.
  • HEK 293T cells were transfected with a plasmid encoding an NF-KB-driven luciferase reporter. After 24 hours, cells were stimulated for 20 minutes in permeabilization buffer (5 pg/mL digitonin) in the presence of water or 10 pg/mL, 30 pg/mL, or 50 pg/mL of indicated synthetic compounds according to the invention. Treatment was removed; cells were washed and incubated for 6 hours in complete medium. A luciferase assay was then performed. JS-12a control: D-glycero-D-manno heptose. The results are the means ⁇ standard error of the mean of three technical replicates.
  • JS-10 7-0-(Aminoethyl)-D-glycero-3-D-manno-heptopyranose phosphate
  • JS-11 7-0-(Aminoethyl)-D-glycero-D-manno-heptopyranose
  • JS-12 D-glycero-D- manno-heptose
  • Js-13 pH-Rhodo-7-0-(aminoethyl)-D-glycero- -D-manno-heptopyranose phosphate.
  • FIG. 13 Stimulation of human colonic epithelial cells by compounds / products according to the invention.
  • Human colonic epithelial cells HCT 1 16 that were either wild type (WT) or deficient in TIFA protein expression (knockout, TIFA KO) were stimulated for 20 minutes in permeabilization buffer (5 pg/mL digitonin) in the presence of water, 20 pg/mL of the Nodi agonist C12-E-DAP (39.8 pM, which induces inflammation in a TIFA-independent manner) or 30 pM or 150 pM of synthetic compound according to the invention.
  • permeabilization buffer 5 pg/mL digitonin
  • FIG. 14 Effects of compounds / products according to the invention on HEK 293T cells encoding an NF-KB-driven luciferase reporter gene.
  • HEK 293T cells were transfected with a plasmid encoding an NF-KB-driven luciferase reporter. After 24 hours, cells were stimulated for 20 minutes in permeabilization buffer (5 pg/mL digitonin) in the presence of water, or either 30 pM or 150 pM of indicated synthetic compounds according to the invention. Treatment was removed; cells were washed and incubated for 6 hours in complete medium. A luciferase assay was then performed. The results are the mean and standard error of the mean of three technical replicates and are representative of three independent experiments.
  • JS-7 JS-7:D-glycero-p-D-manno-heptose-phosphate; JS-10: 7-0-(Aminoethyl)-D-glycero- -D-manno-heptopyranose phosphate; JS-14: Biotin-7-O-
  • FIG. 15 Stimulation of human macrophages by compounds / products according to the invention.
  • Human macrophage cells THP-1 were stimulated for 20 minutes in permeabilization buffer (5 pg/mL digitonin) in the presence of water, 39.8 pM of the Nodi agonist C12-E-DAP (which stimulates in a TIFA-independent manner), or either 30 pM or 150 pM of synthetic compound according to the invention.
  • Nodi agonist C12- iE-DAP (20pg/ml, 39.8pM); JS-7: JS-7: D-glycero- -D-manno-heptose-phosphate; JS-10: 7-0- (Aminoethyl)-D-glycero- -D-manno-heptopyranose phosphate; JS-14: Biotin-7-0-(aminoethyl)-D- glycero- -D-manno-heptopyranose phosphate.
  • FIG. 16 Stimulation of human colonic epithelial cells by compounds / products according to the invention.
  • Human colonic epithelial cells HCT 1 16 that were either wild type (WT) or deficient in TIFA protein expression (knockout, TIFA KO) were stimulated for 20 minutes in permeabilization buffer (5 pg/mL digitonin) in the presence of water, 20 pg/mL of the Nodi agonist C12-E-DAP (39.8 pM, which induces inflammation in a TIFA-independent manner) or 30 pM or 150 pM of synthetic compound according to the invention.
  • permeabilization buffer 5 pg/mL digitonin
  • JS-24a 7-0-([Biotin]-dPEG4-amidoethyl)-D-Glycero-p-D-manno-heptopyranosyl phosphate
  • JS- 25a 7-0-([Fluorescein]-amidoethyl)-D-Glycero-p-D-manno-heptopyranosyl phosphate
  • Nodi agonist C12-iE-DAP (20pg/ml, 39.8mM).
  • FIG. 17 Effects of compounds / products according to the invention on HEK 293T cells encoding an NF-KB-driven luciferase reporter gene.
  • HEK 293T cells were transfected with a plasmid encoding an NF-KB-driven luciferase reporter. After 24 hours, cells were stimulated for 20 minutes in permeabilization buffer (5 pg/mL digitonin) in the presence of water, TNF-a (80ng/mL) or either 30 pM or 150 pM of indicated synthetic compounds according to the invention. Treatment was removed; cells were washed and incubated for 5 hours in complete medium. A luciferase assay was then performed.
  • JS-24a 7-0-([Biotin]- dPEG4-amidoethyl)-D-Glycero-p-D-manno-heptopyranosyl phosphate; JS-25a: 7-0-
  • CRM cross-reacting material (a genetic mutant of diphtheria toxin); HBP: heptose-1 , 7-biphosphate or D-g/ycero-D-manno-heptopyranose-1 ,7-p-phosphate; HMP- b: D-g/ycero-D-manno-heptopyranose-i p-phosphate (also named heptose-1- monophosphate or JS7); PAMP: pathogen associated molecular pattern; GFP: green fluorescent protein; Sup: supernatant; WT: wild type; TIFA: tumor necrosis factor receptor-associated factor (TRAF)-interacting protein with forkhead-associated domain; TIFA KO: TIFA protein expression knockout.
  • HBP heptose-1 , 7-biphosphate or D-g/ycero-D-manno-heptopyranose-1 ,7-p-phosphate
  • HMP- b D-g/ycer
  • the wording "and/or” is intended to represent an inclusive-or. That is, “X and/or Y” is intended to mean X or Y or both, for example. As a further example, “X, Y, and/or Z” is intended to mean X or Y or Z or any combination thereof.
  • the words“comprising” (and any form of comprising, such as“comprise” and“comprises”), “having” (and any form of having, such as“have” and“has”),“including” (and any form of including, such as“include” and “includes”) or“containing” (and any form of containing, such as “contain” and “contains”), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.
  • the terms“Ci -6 » means a chain containing from 1 to 6 carbon atoms (or more generally x to y carbon atoms), being saturated or unsaturated, linear or branched, as permitted by the laws of organic chemistry, well understood by the person skilled in the art.
  • the term“phosphorylated heptose compound” refers to a monosaccharide with seven carbon atoms, wherein at least one hydroxyl group is replaced by a group comprising a phosphorus atom.
  • the term “mono- phosphorylated heptose compound” refers a monosaccharide with seven carbon atoms, wherein one hydroxyl group is replaced by a group comprising a phosphorus atom.
  • the term also refers to a derivative or an analogue of such compound.
  • label refers to any molecule used in such a way as to attach a functional or diagnostic or other moiety to the molecule, such as but not limited to fluorescent tags, affinity tags, transfer factor, plasmids or lipids, in such a way that the bioactivity can be for example but not limited to followed, targeted, directed or modified or otherwise guided, analyzed or assessed.
  • the term “modulate” in connection with an immune or inflammatory response refers to a qualitative or quantitative alteration in the immune or inflammatory response in a subject.
  • the term “vaccine” or “vaccine composition” refers to a pharmaceutical composition containing an immunogen.
  • the composition may be used for modulating an immune response in a subject.
  • the term also refers to subunit vaccines, i.e. , vaccine compositions containing immunogens which are separate and discrete from a whole organism with which the immunogen is associated in nature.
  • the term “effective amount” refers to the amount of a compound medicinal molecule or reaction product sufficient to cure, alleviate or partially arrest the clinical manifestations of a given disease and its complications in a therapeutic intervention comprising the administration of said compound or reaction product.
  • An effective amount for each purpose will depend on the severity of the disease or injury as well as the weight and general state of the subject.
  • the term“functional molecule” refers to a molecule that is administrable to a mammal for modulating or labeling a least one physiological parameter, marker or target in a cell, organ, biological system, organism or mammal.
  • the methods of the present disclosure may be used to therapeutically or prophylactically treat any subjects for which increased activation of the immune system or an altered immune response would be beneficial.
  • This includes, but is not restricted to a subject suffering from a condition which deleteriously affects the immune system, including any subjects at a heightened risk of infection or actually infected, for example due to surgery or imminent surgery, injury, illness, radiation or chemotherapy, and any subjects suffering from autoimmune diseases, inflammatory disorders, cancers, and diseases which cause the normal metabolic immune response to be compromised, such as HIV (AIDS).
  • HIV HIV
  • prevention and preventing mean the management and care of a subject for the purpose of preventing the occurrence of a condition, such as a disease or disorder.
  • the composition may be used for inducing or enhancing an immune response against a potential pathogen in a subject for blocking infection, delaying onset or decreasing transmission.
  • the subject to be treated is preferably a mammal, in particular a human being.
  • treatment and“treating” mean the management and care of a subject for the purpose of combating a condition, such as a disease or disorder.
  • the term is intended to include the full spectrum of treatments for a given condition from which the patient is suffering, such administration of the active compounds to alleviate the symptoms or complications, to delay the progression of the condition, and/or to cure or eliminate the condition.
  • the subject to be treated is preferably a mammal, in particular a human being.
  • the term “subject” is understood as being any mammal including a human being treated with a compound of the invention.
  • HMP b-D-heptose-l -phosphate
  • HMP b-D-heptose-l -phosphate
  • the synthesis of the HMP is significantly simpler and likely more commercially realisable than that of the HBP.
  • this creates the potential opportunity to modify the 7-position of the HMP to link this immunomodulator to a huge variety of functional molecules whose processing and activity may be controlled and directed by conjugation to this PAMP-like molecule.
  • the inventors have identified phosphorylated heptose-derivatives useful for conjugating with a functional molecule and modulating their immunomodulatory activities thereof. These conjugates may be useful in modulating (i.e. enhancing or decreasing) an immune response in a subject or labeling the molecule in such a way that it may be followed, targeted, directed or modified or otherwise guided.
  • the resulting HMP-conjugate is capable of modulating an immune response in a subject.
  • R is selected from the group consisting of: -C(0)0H, -C(0)H, -Co- 6 alkyl-C(0)-Ci- 6 alkyl, -Co- 6 alkyl-N 3 , -Ci -6 alkyl, -C 0- 6 alkyl -0-Ci- 6 alkyl, -Co- 6 alkyl-aryl, -Co- 6 alkyl-0-aryl, -Co-6alkyl-C2-6-allyl groups, and -C 0- 6 alkyl-0-C 2-6 -allyl, wherein said -Co- 6 alkyl-C(0)-Ci-6 alkyl, -Ci -6 alkyl, C 0 -6alkyl-O-Ci- 6 alkoxy, -aryl, C 0 - ealkyl-O-aryl, Co 6 alkyl-C 2 e-allyl groups, and Co-6alkyl-0-
  • R is selected from the group consisting of: -Ci -6 alkyl-0P0 3 C(0)-Ci- 6 alkyl, -Ci -6 alkyl- OPO3C 1 -6 alkyl, -C 1 -6 alkyl-OP03-Ci-6-0-alkoxy, -C 1 -6 alkyl-0P03-aryl, -C 1 -6 alkyl-OPC>3-C 2 - 6 -allyl,
  • -Ci -6 alkyl-0P0 3 C(0)-Ci- 6 alkyl, -Ci -6 alkyl-0P0 3 Ci- 6 alkyl, -Ci -6 alkyl- OP0 3 -Ci- 6 -0-alkoxy, -Ci -6 alkyl-0P0 3 -aryl, -Ci -6 alkyl-0P0 3 -C 2-6 -allyl group is optionally substituted with a: amino, acyloxy, alkoxy, carboxyl, carbalkoxyl, hydroxy, trifluoromethyl, cyano, nitro, acyl, or a halo group;
  • R is selected from the group consisting of: -C(0)0H, -C(0)H, -C(0)-Ci-6 alkyl, - N3, -C 1 -6 alkyl, -O-C 1 -6 alkyl, -aryl, -O- aryl, -C 2-6 -allyl groups, and -0-C 2-6 -allyl, wherein said -C(0)-Ci -6 alkyl, -Ci -6 alkyl, -0-Ci -6 alkoxy, -aryl, -O-aryl, -C 2-6 -allyl groups, and -0-C 2-6 -allyl is optionally substituted with : amino, acyloxy, alkoxy, carboxyl, carbalkoxyl, hydroxy, trifluoromethyl, cyano, nitro, acyl, or a halo group;
  • R is selected from the group consisting of: CH 2 0P0 3 C(0)-CI- 6 alkyl, CH 2 0P0 3 Ci- 6 alkyl, CH 2 0P0 3 -CI- 6 -0 alkoxy, CH 2 0P0 3 -aryl, CH 2 0P0 3 -C 2-6 -allyl, wherein said CH 2 0P0 3 C(0)-CI- 6 alkyl, CH 2 0P0 3 Ci- 6 alkyl, CH 2 0P0 3 -Ci- 6 -0 alkoxy, CH 2 0P0 3 -aryl, CH 2 0P0 3 -C 2-6 -allyl group is optionally substituted with a: amino, acyloxy, alkoxy, carboxyl, carbalkoxyl, hydroxy, trifluoromethyl, cyano, nitro, acyl, or a halo group; or a salt thereof.
  • R is selected from the group consisting of: -Ci -6 alkyl, -C(0)0H, -C(0)H, -C(0)-Ci -6 alkyl, -Co- 6 alkyl-0-Ci- 6 alkyl, -C 0- 6a I ky l-C 2 -6- a I ly I groups, and -0-C 2-6 -allyl, wherein each of said -Ci_6alkyl, -C(0)-Ci-6 alkyl, -Co- 6 alkyl-0-Ci- 6 alkyl, _-Co- 6 alkyl-C 2-6 -allyl, and -Co- 6 alkyl-0-C 2-6 -allyl group is optionally substituted with a : amino, alkoxy, hydroxy, carboxy or nitro group;
  • R is selected from the group consisting of: CH 2 0P0 3 C(0)-CI- 6 alkyl, CH 2 0P0 3 Ci- 6 alkyl, CH 2 0P0 3 -CI- 6 -0 alkoxy, CH 2 0P0 3 -aryl, CH 2 0P0 3 -C 2-6 -allyl, wherein each of said CH 2 0P0 3 C(0)-CI- 6 alkyl, CH 2 0P0 3 Ci- 6 alkyl, CH 2 0P0 3 -Ci- 6 -0 alkoxy, CH 2 0P0 3 -aryl, CH 2 0P0 3 -C 2-6 -allyl group is optionally substituted with a: amino, alkoxy, hydroxy, or nitro group; or a salt thereof.
  • R is a linkage moiety for conjugating with a functional molecule.
  • the resulting HMP-conjugate is capable of modulating an immune response in a subject.
  • R is selected from the group consisting of: -C(0)-Ci-6 alkyl, - N3, -C 1 -6 alkyl, -O-C 1 -6 alkyl, -aryl, -O-aryl, -C 2 -6-allyl groups, and -0-C 2-6 -allyl, wherein each of said -C(0)-Ci- 6 alkyl, -Ci -6 alkyl, -0-Ci- 6 alkoxy, -aryl, - O-aryl, -C 2-6 -allyl, and -0-C 2-6 -allyl groups is optionally substituted with a : amino, alkoxy, hydroxy, or nitro group; or R is selected from the group consisting of: CH 2 0P0 3 C(0)-CI- 6 alkyl, CH 2 0P0 3 CI- 6 alkyl, CH 2 0P0 3 -CI- 6 -0 alkoxy,
  • (la) is substituted with an“R” group that may be selected from the group consisting of: - C(0)0H, -C(0)H, -C(0)Ci- 6 alkyl, -CH 2 N 3 , -CH 2 OCI- 6 alkyl, -CH 2 Oaryl, and -CH 2 OC 2.6 - allyl groups, wherein said -C(0)Ci- 6 alkyl, -CH 2 OCI- 6 alkyl, -CH 2 Oaryl, and -CH 2 OC 2.6 - allyl group is optionally substituted with a : amino, acyloxy, alkoxy, carboxyl, carbalkoxyl, hydroxy, trifluoromethyl, cyano, nitro, acyl, or a halo group; or a salt thereof. More particularly, the group R is: CH 2 O-CH 2 CH 2 -NH 2 ; -C(0)OH, -C(0)H, -C(0)NH 2 or -CH 2 N 3
  • HMP via a phosphate residue, wherein R is CH 2 0P0 3 C(0)-CI- 6 alkyl, CH 2 0P0 3 Ci- 6 alkyl, CH 2 0P0 3 -Ci- 6 -0 alkoxy, CH 2 0P0 3 -aryl, CH 2 OPC>3-C 2 -6-allyl.
  • CH 2 0PC>3C(0)-CI-6 alkyl, CH 2 OPC>3CI-6 alkyl, CH 2 0P0 3 -CI- 6 -0 alkoxy, CH 2 0P0 3 -aryl, CH 2 0P0 3 -C 2-6 -allyl groups is optionally substituted with a: amino, acyloxy, alkoxy, carboxyl, carbalkoxyl (alkoxycarbonyl), hydroxy, trifluoromethyl, cyano, nitro, acyl, or a halo group; or a salt thereof.
  • R is selected from the group consisting of: Ci- 6 alkyl-NH 2 ; - Co-6 alkyl-C-O-Co-6 alkyl-NH 2 , -C(0)0H, -C(0)H, -C 0-6 alkyl-C(O)C 0-6 alkyl-NH 2 , C 0-6 alkyl- N 3 .
  • R is selected from the group consisting of:CH 2 -NH 2 ; -C(0)0H, - C(0)H, -C(0)NH 2 and - N 3 .
  • R is selected from the group consisting of: CH 2 -NH 2 ; -
  • the HMP-linker may be chosen from:
  • conjugate-compounds can now be targeted to the unique arm of the immune system that processes HMP / HBP, thus potentially enhancing the immunological impact of the conjugated functional molecule.
  • a compound comprising a functional molecule conjugated to a D-glycero-D-manno- heptopyranose I b-phosphate (heptopyranose-1 -monophosphate; HMP) moiety.
  • the functional molecule is conjugated at position-7 of HMP, or -2 of HMP.
  • the functional molecule is conjugated at position-7 of HMP.
  • the invention provides a conjugate compound of formula (II) as defined herein, for use in targeting and/or modulating an immune response in a subject.
  • the conjugate-compound is defined by formula (I la):
  • the HMP-linker is conjugated to a functional molecule for modulating a host’s immune response to this functional molecule.
  • the functional molecule is a protein (e.g. carrier proteins such as: CRM, protein D, or TT), polypeptide (e.g. Ova-specific peptide: CGGSIINFEKLTEWTSSNVMEER (SEQ ID No.1)), an antibody, a lipid (e.g. glycerolipid), a fatty acid (e.g. stearic acid or palmitic acid), a carbohydrate, a drug, a fluorescent tag, an affinity tag or a transfer factor molecule.
  • the functional molecule is an antigen, a biological response modifier (BRM, such as a hormone, immunogen, antibody, etc.) or a marker.
  • BRM biological response modifier
  • the functional molecule is a drug such as, for example, an anti- HIV drug including, but not limited to: Nucleoside/Nucleotide reverse transcriptase inhibitors (NRTI), such as for example, abacavir (ziagen), efavirenz/emtriacitabine/tenofovir disoproxil fumarate (atripla), lamivudine/zidovudine (combivir), emtriacitabine/rilpivirine/tenofovir disoproxil fumarate (complera), emtricitabine (emtriva), lamivudine (Epivir), abacavir/lamivudine (Epzicom), zidovudine (Retrovir), abacavir/lamivudine/zidovudine (Trizivir), emtricitabine/tenofovire disoproxil fumarate (Truvada), didanosine (Videx),
  • NRTI Nu
  • Some anti-HCV drugs such as, for example: protease inhibitors (PI) approved for hepatitis C only are sometimes prescribed by doctors for HIV: simeprevir (Olysio), boceprevir (Victrelis), ombitasvir/paritaprevir/ritonavir/dasabuvir (Viekira Pak).
  • PI protease inhibitors
  • the functional molecule that could be linked to HMP may include anti-cancer drugs, such as, but not limited to: Abiraterone, Alemtuzumab, Anastrozole, Aprepitant, Arsenic trioxide, Atezolizumab, Azacitidine, Bevacizumab, Bleomycin, Bortezomib, Cabazitaxel, Capecitabine, Carboplatin, Cetuximab, Chemotherapy drug combinations, Cisplatin, Crizotinib, Cyclophosphamide, Cytarabine, Denosumab, Docetaxel, Doxorubicin, Eribulin, Erlotinib, Etoposide, Everolimus, Exemestane, Filgrastim, Fluorouracil, Fulvestrant, Gemcitabine, HPV Vaccine, Imatinib, Imiquimod, Ipilimumab, Ixabepilone, Lapatinib, Lenalidom
  • the functional molecule is an antigen, more particularly a molecule that targets a cell differentiation antigen, such as a-specific binding partner for targeting the HMP moiety to a particular type of cells, be it cancer cells, immune cells, or cells of any particular organ or location in the body.
  • a cell differentiation antigen such as a-specific binding partner for targeting the HMP moiety to a particular type of cells, be it cancer cells, immune cells, or cells of any particular organ or location in the body.
  • conjugated functional molecule is a molecule that targets a cell differentiation antigen, such as a specific binding partner
  • this molecule may be used as a“magic bullet” to target the immunomodulatory HMP moiety to a particular type of cells, such as, for example, HIV reservoir cells.
  • the HMP moiety may be cleaved from the conjugate to release the HMP moiety to the targeted cell type.
  • the person skilled in the art will adapt the linkage-moiety to be used for cleavage to be carried out once the conjugate has reached the appropriate cell type and release the HMP immunomodulatory moiety.
  • the cleavable linker can have cleavable unit that is a photocleavable, enzyme-cleavable or chemically- cleavable unit.
  • the cleavable linker can have a cleavable unit such as a disulfide (chemically cleavable), nitrobenzo (a photocleavable unit), or amine, amide or ester (enzyme-sensitive cleavable units).
  • the cleavable linker can be of the formula: X - s - S - Z, or, more
  • the functional molecule conjugated to HMP is an immunogen, particularly an immunogen expressed by, or derived from, an infectious agent, such as for example an infectious bacterial, viral or parasitic pathogens, including Gram-negative bacterial pathogens belonging to the genus Neisseria (including Neisseria meningitidis, Neisseria gonorrohoeae), Escherichia (including Escherichia coli), Klebsiella (including Klebsiella pneumoniae), Salmonella (including Salmonella typhimurium), Shigella (including Shigella dysenteriae, Shigella flexneri, Shigella sonner), Vibrio (including Vibrio cholerae), Helicobacter (including Helicobacter pylori), Pseudomonas (including Pseudomonas aeruginosa), Burkholderia (including Burkholderia multivorans), Haemophilus (including Ha
  • Immunogens may also be derived from pathogenic viruses including Adenoviridae (including Adenovirus), Herpesviridae (including Epstein-Barr virus, Herpes Simplex Viruses, Cytomegalovirus, or Varicella Zoster virus), Papillomviridae, Poxviridae (including Papillomavirus), Hepadnaviridae (including Hepatitis B virus), Parvoviridae, Astroviridae, Caliciviridae, Picornaviridae (including Coxsackievirus, Hepatitis A virus, Poliovirus), Coronaviridae, Flaviviridae (including Hepatitis C virus, Dengue virus), Togaviridae (including Rubella virus), Hepeviridae, Retroviridae (including HIV), Orthomyxoviridae (including influenza virus, Arenaviridae, Bunyaviridae, Filoviridae, Paramyxoviridae (including Meas), and Me
  • Immunogens may also be derived from pathogenic fungal infections including those caused by Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis, or Coccidioides. Vaccines may also target parasitic pathogens including Leishmania, Plasmodium, Toxoplasma, Trypanosoma and Schistosoma.
  • the immunogen may be derived from a protein or other molecules expressed on or within the subject's own cells, such as a tumor-specific immunogen or cancer- specific immunogen, to stimulate an immune response against the pathogenic cells or tissues.
  • the HBP may be introduced directly into a tumor to increase the immune response against the tumor.
  • the HMP-immunogen conjugate of the invention can be administered as part of a vaccine formulation.
  • the linker of the invention is conjugated to a marker, such as, for example, a label or a tag.
  • a marker such as, for example, a label or a tag.
  • the marker or tag is chosen from fluorescent and / or enzymatic tags, such as but not limited to: biotin, rhodamine, fluorescein, CF tags, EvoBlue, rhodopsin, GFP, fluorescent red, fluorescent orange biotin or other labels such as, for example:
  • linkers are synthesized to understand how the nature of the linker may impact the biological activity. With this knowledge, a vast array of functional molecules are appropriately attached to an HMP-linker to examine several aspects of this PAMP’s interaction with the immune system and to augment and specifically target the linked entities processing within the immune system.
  • These can include but are not limited to, the identification of the receptor for HMP, an understanding of the processing of HMP, developing antibodies to HMP to follow the trafficking of the molecule, or targeting HMP to certain cells within the immune system by linking to a fluorescent moiety for example.
  • immunomodulatory activity the method comprising the step of: conjugating a functional molecule with a compound of formula (I) or (la) as defined herein.
  • conjugates of the present invention can be synthesized via the synthesis of HMP that is significantly simpler and likely more commercially realisable than that of HBP.
  • a conjugate compound of formula (II) or (lla) as defined herein for use in targeting and/or modulating an immune response in a subject.
  • the ability to target the modulation of the immune response of the subject is useful for preventing, treating, ameliorating, or inhibiting an injury, disease, disorder or condition wherein the modulation of the immune response is beneficial.
  • the conjugate compound for use is carried out in the context of a vaccine for the treatment or prevention of disease.
  • a conjugate also called a compound of formula (II) or (lla) as defined herein, for targeting and/or modulating an immune response in a subject.
  • the modulation of the immune response of the subject is useful for preventing, treating, ameliorating, or inhibiting an injury, disease, disorder or condition wherein the modulation of the immune response is beneficial.
  • the use of the compound is carried out in the context of a vaccine for the treatment or prevention of disease.
  • the use of the compound of formula (II) or (lla as defined herein is carried out for the manufacture of a medicament for modulating an immune response in a subject.
  • the immune response of the subject is enhanced.
  • the molecule conjugated to the HMP-linker is an antigen or an immunogen, more particularly chosen from cell-differentiation antigen, or antigens from cancer cells, bacteria, viruses or parasites.
  • the immunogen used for embodying the invention is from Gram-negative bacteria, most particularly chosen from: Neisseria, Escherichia, Klebsiella, Salmonella, Shigella, Vibrio, Helicobacter, Pseudomonas, Burkholderia, Haemophilus, Moraxella, Bordetella, Francisella, Pasteurella, Borrelia, Campylobacter, Yersinia, Rickettsia, Treponema, Chlamydia and Brucella.
  • Gram-negative bacteria most particularly chosen from: Neisseria, Escherichia, Klebsiella, Salmonella, Shigella, Vibrio, Helicobacter, Pseudomonas, Burkholderia, Haemophilus, Moraxella, Bordetella, Francisella, Pasteurella, Borrelia, Campylobacter, Yersinia, Rickettsia, Treponema, Chlamydia and Brucella.
  • the immunogen useful for the invention is from Grampositive bacteria, most particularly chosen from: Staphylococcus, Streptococcus, Listeria, Corynebacterium, Enterococcus, Clostridium and Mycobacterium.
  • the immunogen used for embodying the present invention is derived, or comes, from a virus, more particularly chosen from: Adenoviridae, Herpesviridae, Papillomviridae, Poxviridae, Hepadnaviridae, Parvoviridae, Astroviridae, Caliciviridae, Picornaviridae, Coronaviridae, Flaviviridae, Togaviridae, Hepeviridae, Retroviridae, Orthomyxoviridae, Arenaviridae, Bunyaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, Reoviridae and Human Immunodeficiency (HIV) viruses.
  • Adenoviridae Herpesviridae, Papillomviridae, Poxviridae, Hepadnaviridae, Parvoviridae, Astroviridae, Caliciviridae, Picornavirid
  • the immunogen is from a parasite, more particularly chosen from: Leishmania, Plasmodium, Toxoplasma, Trypanosoma and Schistosoma.
  • the immunogen may originate from a cancer-specific antigen, more particularly some or all of the cancer-specific antigens listed in Table 1 : Table 1 (cancer-specific antigen useful for conjugating with HMP-linker
  • Idiotypic Ig TCR B, T leukemia, lymphoma, myeloma
  • BRCA breast cancer antigen
  • CDK4 cyclin-dependent kinase-4
  • CEA carcino-embryonic antigen
  • CML66 chronic myelogenous leukemia (antigen) 66
  • CT cancer testis
  • HPV human papilloma virus
  • Ep-CAM epithelial cell adhesion molecule
  • Ig immunoglobulin
  • MART-1/-2 melanoma antigen recognized by T cells- 1/-2
  • MC 1R melanocortin-1 -receptor
  • SAP- 1 stomach cancer- associated protein tyrosine phosphatase- 1
  • TAG-72 tumor antigen-72
  • TCR T cell receptor
  • TGF-PRII transforming growth factor-b receptor II
  • TRP tyrosinase-related protein.
  • the conjugate compound of the invention wherein the immune response of the subject is decreased, more particularly in the case where the immune response is provoked by an inflammatory response or an allergic reaction to a foreign element.
  • the compound of the invention is conjugated with an immune-inhibitory molecule.
  • a method for modulating an immune response in a subject comprising administering an effective amount of the compound of formula (II) or (I la) as defined herein to a subject suffering therefrom.
  • the modulation of immune response is necessary (or indicated or prescribed by a treating physician) for increasing an immune response to an infection caused by: bacteria, virus, or parasites, such as those defined herein above.
  • the modulation of immune response is necessary (or indicated or prescribed by a treating physician) for decreasing an immune response such as inflammation or an allergic reaction in a subject.
  • the subject is a human.
  • a method for the treatment of HIV comprising the steps of: administering an HMP-conjugated to an anti- HIV molecule, wherein the HMP moiety stimulates HIV latently-infected cells to start producing virus, and wherein the anti-HIV molecule kills the produced virus; whereby the HMP-anti-HIV conjugate eradicates the latent viral pool and treats the HIV infection.
  • a method for the treatment of HIV comprising the steps of: administering an HMP-conjugated to an HIV- reservoir cell-specific antigen binding partner to a subject, and treating said subject with an anti-HIV drug, wherein the conjugate-HMP moiety stimulates HIV latently-infected cells to start producing virus, and the anti-HIV drug kills the produced virus; whereby the HMP- anti-HIV conjugate eradicates the latent viral pool and treats the HIV infection.
  • the anti-HIV molecule or drug is chosen from: Nucleoside/Nucleotide reverse transcriptase inhibitors (NNRTI), Multiclass inhibitors, Non-nucleoside reverse transcriptase inhibitors (NNRTIs), Protease inhibitors (PI), Entry inhibitors, Chemokine co-receptor antagonists (CCR5 antagonists), Cytochrome P4503A (CYP3A) inhibitors. More particularly, the anti-HIV drug is as defined herein above.
  • JS12a refers to D-glycero-D-manno heptose control.
  • Nitrile 17 (196 mg, 479 pmol) was coevaporated with toluene and then dissolved in THF (5 mL). To the mixture was added slowly borane-dimethyl sulfide (182 mI_, 1.9 mmol). After 3 hours at 80°C, the solution was cooled at 0 °C and methanol was slowly added (5 mL). After 16 hours, the solution was concentrated in vacuo and the compound was dissolved in dioxane (1.3 mL) and a NaHC0 3 (sat. aq.) solution (2.6 mL).
  • JS12a was synthesized as described by Brimacombe et al. [9, 10] pH-Rhodo-7-0-(aminoethyl)-D-glycero ⁇ -D-manno-heptopyranose phosphate (JS13a)
  • JS10a (700 pg) was dissolved in PBS x 1 (100 mI_) and pH-Rhodo activated ester (1 mg in 100 mI_ DMSO) was added to the mixture. After 24 hours, the DMSO the mixture was concentrated in vacuo and JS12a was purified using reverse phase. LRMS and HPLC showed that JS13a was only present at a ratio of 6% in the mixture and was tested according to this.
  • JS10a (4.1 mg, 12 mhioI) was dissolved in water (1 mL) and Biotin-dPEG4-
  • Additional compounds (JS 15 to JS19) can be synthesized according to standard techniques such as those described in Li et al., Carbohydrate Research, 2016, 432, 71-75 and Li et al. Organic letters, 2014, 16, 5628-5631.
  • the R group may be any linkage moiety for conjugating with a molecule, and the resulting conjugate compound is capable of targeting and/or modulating an immune response thereof.
  • the R group can be selected from the group consisting of: -C(0)OH, -C(0)H, -Co- 6 alkyl-C(0)-Ci- 6 alkyl, -C 0-6 alkyl-N 3 , -Ci- 6 alkyl, -C 0-6 alkyl -0-Ci- 6 alkyl, -C 0-6 alkyl-aryl, -Co- 6 alkyl-0-aryl, -Co- 6 alkyl-C2-6-allyl groups, and -Co- 6 alkyl-0-C 2-6 - allyl, wherein said -C 0-6 alkyl-C(O)-Ci-6 alkyl, -Ci -6 alkyl, C 0 -6alkyl-O-Ci- 6 alkoxy, -aryl, C 0- 6 alkyl-0-aryl, C 0-6 alkyl-C2 b-allyl groups, and Co- 6 alkyl-
  • the R group can be selected from the group consisting of: - CH 2 -NH 2 ; -C(0)0H; -C(0)H; - N 3 ; - lower alkyl or -O-lower alkyl groups containing 1-6 saturated or unsaturated carbon atoms; and -aryl, -O-aryl, -O-allyl or -allyl groups having 2-6 carbon atoms; wherein such alkyl, -O-alkyl, aryl, -O-aryl, -allyl or -O-allyl groups may be unsubstituted or may be substituted with : halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl, or amino groups; or with a hydroxy, an amino or a halo group.
  • HMP via a phosphate residue, wherein R is C0PC>3C(0) Ci-6 alkyl, -COPO3C1-6 alkyl, -COPO3- C1-6-O alkoxy, -COPCb-aryl, - COP0 3 -aryl, -C0P0 3 -C 2-6 -allyl.
  • R is C0PC>3C(0) Ci-6 alkyl, -COPO3C1-6 alkyl, -COPO3- C1-6-O alkoxy, -COPCb-aryl, - COP0 3 -aryl, -C0P0 3 -C 2-6 -allyl.
  • the compounds of formula (I) as defined herein can be synthesized according to standard techniques, particularly according to US patent 5,856,462.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Virology (AREA)
  • Organic Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pulmonology (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • AIDS & HIV (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Heptose-1-monophosphate-7-derivatives are modifiable immunomodulators that can be used to prepare clinically active conjugate compounds. Such conjugate compounds are useful in modulating an immune response in a subject.

Description

D-GLYCERO-B-D-HEPTOSE 1 -PHOSPHATE (HMP) CONJUGATES AND USE FOR TARGETED IMMUNE MODULATION
FIELD
[0001] The present invention relates generally to phosphorylated heptose compounds. More specifically, the present invention relates to use of heptopyranose phosphate and conjugates thereof in modulating, or targeting the modulation of, an immune response in a subject, to improve the specificity and effect of HMP and enhance protection.
BACKGROUND
[0002] The immune system provides protection against infectious agents, including bacteria, viruses, fungi, and parasites. A substantial number of medical conditions are associated with a compromised immune system and its associated increase in susceptibility to infectious agents. Thus, for example, patients undergoing surgery, radiation or chemotherapy, and those suffering from autoimmune diseases and diseases interfering with a normal metabolic immune response, such as HIV (AIDS), are all at a heightened risk of developing pathological conditions resulting from infection. While pharmaceuticals - antibiotics, such as ampicillin, tetracycline and quinolones, for example, in the case of bacterial infections - offer treatment options, resistance of the infectious agent to these pharmaceuticals is an increasingly significant concern. [0003] Therefore, there is need for immune activating or modulating strategies to induce responses better able to prevent or combat infection. Furthermore, vaccines preventing or treating infection by many microbial organisms have been developed, however there is an ongoing need for additional vaccine formulations, as the immune stimulatory profile of known vaccine formulations is frequently suboptimal. [0004] Vaccines have also been proposed to help the immune system target cancerous cells or tissues, however there is a need to improve the immune response so that it can more effectively combat the cancer. Finally, there is ongoing need to alter pathogenic immune responses, and particularly pathogenic inflammatory responses, to reduce disease symptoms and/or progression. [0005] The ability to modulate the immune system of the host is becoming more and more critical as we strive to improve the immune response of individuals to generate a protective response. This is outlined for example in WO 2016/054745 entitled“Methods of modulating immune system responses.” Understanding and exploiting potential new immunomodulation pathways are also critical to the development of new drugs.
[0006] Pathogen-associated molecular patterns (PAMPs; also known as microbial- associated molecular patterns, or MAMPs) are molecules produced by microbes that are specifically recognised and used as cues by the human immune system to generate innate and adaptive immune responses to keep foreign pathogens at bay. The ability to synthesise PAMPs will enable the specific modulation of the immune system to improve the immune response and generate protection.
[0007] Only a limited number of PAMPs have been identified, e.g. lipopolysaccharide (LPS), unmethylated CpG motifs within bacterial DNA, and flagellin. This limits the opportunity to investigate the immunomodulatory properties of these molecules. In most cases, PAMPs are difficult to synthesise completely or isolate at required and reproducible levels of purity, and thus precludes an opportunity to specifically address how these PAMPs interact with the immune system to exploit this relationship, when a pure, fully characterised supply of the PAMPs is unavailable.
[0008] Therefore, there is a need in the art to develop further PAMP molecules as treatment and prevention options against infections caused by infectious agents, cancerous cells and immune or inflammatory diseases.
SUMMARY
[0009] The inventors have identified phosphorylated heptose compounds (such as HMP) as PAMP-like molecules useful in modulating an immune response in a subject.
[0010] More specifically, in accordance with a first aspect of the invention, there is provided an HMP-linker corresponding to formula (I):
wherein group « R » is a linkage moiety adapted (or suitable) for conjugating with a functional molecule to form a conjugate, and Y is H or an appropriate atom for forming a salt thereof. Particularly, the conjugate is adapted (or capable) of modulating an immune response in a subject. More particularly, the conjugate is adapted (or capable) of targeting a modulation of an immune response in a subject.
[0011] Alternatively, the present invention provides an immunomodulatory compound comprising a molecule conjugated to a D-glycero-D-manno-heptopyranose I b-phosphate (heptopyranose-1-monophosphate; HMP) moiety. Particularly, the molecule is a functional molecule, such as, for example, a drug, a cell surface antigen, a biological response modifier (BRM), a cell differentiation antigen specific binding partner or a marker. More particularly, the functional molecule is conjugated at position-7 of HMP or at position-2 of HMP, most particularly at position-7. [0012] In accordance with a particular aspect, the invention provides a method to prepare an immunomodulator conjugate comprising the step of: conjugating a functional molecule with a compound of formula (I)
whereby the immunomodulator conjugate has immunomodulatory and/or targeting activity.
[0013] In accordance with an alternative aspect, the invention provides a conjugate compound of formula (II):
wherein X is a functional molecule and Y is H or an appropriate atom for forming a salt thereof, for use in targeting and/or modulating an immune response in a subject.
[0014] In accordance with a particular aspect, the use for modulating an immune response in a subject may comprise: a) improving the delivery of this HMP moiety into the cell. This could be achieved by linking the HMP moiety to a lipid or fatty acid or other molecule that would improve the permeability of HMP across cell membranes; b) treating an HIV infection by stimulating HIV latently-infected cells to start producing virus, and killing the produced virus; whereby the HMP-anti-HIV conjugate eradicates the latent viral pool and treats the HIV infection; or c)
[0015] In accordance with an alternative aspect, the invention provides a use of an effective amount of a compound of formula (II) as defined herein, for targeting and/or modulating an immune response in a subject.
[0016] Alternatively, the invention provides a use of a conjugate-compound of formula (II) for the manufacture of a medicament for targeting and/or modulating an immune response in a subject.
[0017] In accordance with an alternative aspect, the invention provides a method for modulating an immune response in a subject, comprising administering an effective amount of the conjugate-compound of formula (II) as defined herein.
[0018] In accordance with an alternative aspect, the invention provides a method for improving the delivery of this HMP moiety into the cell. This could be achieved by linking the HMP moiety to a lipid or fatty acid or other molecule that would improve the permeability of HMP across cell membranes
[0019] In accordance with a particular aspect, there is provided a method for the treatment of HIV comprising the steps of: administering an HMP-derivative conjugated to an anti-HIV molecule, wherein the HMP moiety stimulates HIV latently-infected cells to start producing virus, and wherein the anti-HIV molecule kills the produced virus; whereby the HMP-anti-HIV conjugate eradicates the latent viral pool and treats the HIV infection.
[0020] In accordance with a particular aspect, there is provided a method for treating latent HIV infection comprising the steps of: administering an HMP-derivative conjugated to an HIV-reservoir cell-specific targeting molecule to a subject, and treating said subject with an anti-HIV drug, wherein the HMP moiety stimulates HIV latently-infected cells to produce virus, and the anti-HIV drug kills the produced virus; whereby the method eradicates the latent HIV viral pool.
[0021] In accordance with a further aspect, the invention provides a method for synthesizing a compound of formula (II) comprising the steps of Scheme 1 (Figure 1A): Scheme 1.
a) protection of hydroxy group of compound 7; b) hydroxylation of double bond of compound 16; c) reduction of compound 17; d) protection with protecting group, cleavage of acetonide and protecting group and acetylation gave acetylated compound 21 ; e) deprotection of anomeric acetyl; f) phosphorylation; g) deprotection to form JS10a; and i or j) conjugation to a functional molecule X-activated ester to yield a molecule (I la) which is a particular embodiment of the compound of formula (II). [0022] Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
DESCRIPTION
Brief description of the drawings
[0023] In the appended drawings: [0024] Figure 1A. Reaction scheme 1 for the general synthesis of derivative linkers of HMR-b.
[0025] Figure 1 B. Reaction scheme 2 for the synthesis of particular embodiments of derivative linkers of HMR-b.
[0026] Figures 2A-B: A) 1H NMR spectra of 7-0-(Aminoethyl)-D-Glycero^-D-manno- heptopyranosyl phosphate (JS10); B) HSQC 1H-13C, NMR spectra of compound JS10.
[0027] Figures 3A-B. A) 1H NMR spectra of 7-0-(Aminoethyl)-D-Glycero-D-manno- heptopyranose (JS1 1 a). B) 13C NMR spectra of 7-0-(Aminoethyl)-D-Glycero-D-manno- heptopyranose (JS1 1 a).
[0028] Figures 4A-B. A) 1H NMR spectra of D-glycero-D-manno heptose (JS12a). B) 13C NMR spectra of D-glycero-D-manno heptose (JS12a).
[0029] Figures 5A-C. A) 1H NMR spectra of 7-0-(Aminoethyl-pH-Rhodo)-D-Glycero- b-D-manno-heptopyranosyl phosphate (JS13a). B) LMRS spectra of 7-0-(Aminoethyl-pH- Rhodo)-D-Glycero^-D-manno-heptopyranosyl phosphate (JS13a). C) HPLC chromatogram of 7-0-(Aminoethyl-pH-Rhodo)-D-Glycero^-D-manno-heptopyranosyl phosphate (JS13a).
[0030] Figures 6A-B. A) 1H NMR spectra of 7-0-([Biotin]amidoethyl)-D-Glycero^-D- manno-heptopyranosyl phosphate (JS14a). B) 13C NMR spectra of 7-0- ([Biotin]amidoethyl)-D-Glycero-b-D-manno-heptopyranosyl phosphate (JS14a).
[0031] Figure 7. 1H NMR spectra of 7-0-([Biotin]-dPEG4-amidoethyl)-D-Glycero^-D- manno-heptopyranosyl phosphate (JS24a)
[0032] Figure 8. 1H NMR spectra of 7-0-([Fluorescein]-amidoethyl)-D-Glycero^-D- manno-heptopyranosyl phosphate (JS25a)
[0033] Figures 9A-B. Inflammatory response of human colonic epithelial cells (HCT 1 16) exposed to chemically synthesized analogues. Wild type (WT) or TIFA knockout (TIFA KO) cells were treated with compounds in the presence of digitonin for 20 min, treatment was removed, cells were washed and then cultured in normal medium without the synthetic compounds for 6 hr before IL-8 levels in culture supernatants were measured by ELISA. (A) Cells were treated with water or 10 pg/mL of the indicated compound. Data are means ± standard error mean of technical triplicates. (B) Cells were treated with the NOD1 agonist C12-iE-DAP (39.8 mM) or with two different molar concentrations of compounds (30 mM or 150 mM). Two batches of dd-b-HBR and dd-b-HMR synthesized independently of one another were tested (batch #1 and batch #2) to assess consistency of the chemical synthesis. Data are means ± standard error mean of two (150 mM treatment) or three (all other treatments) independent experiments. Nodi agonist: Cl2-iE- DAP (20pg/ml, 39.8mM); JS-12: D-glycero-D-manno-heptose; JS-2: D-glycero-D-manno-heptose 7- phosphate; JS-7:D-glycero- -D-manno-heptose-phosphate; JS-8: d-glycero-a-d-manno-heptose- phosphate; JS-9: b-d-mannose phosphate; JS-6:d-glycero- -d-manno heptose 1 ,7-biphosphate; JS-5: d-glycero-a-d-manno heptose 1 ,7-biphosphate.
[0034] Figures 10A-B. Effect of chemically synthesised analogues on human embryonic kidney cells (HEK 293T) encoding an NF-KB-driven luciferase reporter gene. NF-kB luciferase activity was measured in wild type (WT) or TIFA knockout (TIFA KO) cells treated with compounds in the presence of digitonin for 20 min, washed, and then cultured in normal medium without the synthetic compounds for 6 hrs after treatment, before luciferase activity was measured. (A) Cells were treated with 10 pg/mL of the indicated compound. Data are means ± standard error mean of technical triplicates. (B) Cells were treated with two different molar concentrations of compound (30 mM or 150 mM). Two batches of DD-b-HBR and DD-b-HMR synthesized independently of one another were tested (batch #1 and batch #2) to assess consistency of the chemical synthesis. Data are means ± standard error mean of technical triplicates and are representative of three independent experiments. JS-12: D-glycero-D-manno-heptose; JS-2: D-glycero-D-manno-heptose 7-phosphate; JS-7:D-glycero- -D-manno-heptose-phosphate; JS-8: D- glycero-a-D-manno-heptose-phosphate; JS-9: b-D-mannose phosphate; JS-6:D-glycero-3-D- manno heptose 1 ,7-biphosphate; JS-5: D-glycero-a-D-manno heptose 1 ,7-biphosphate.
[0035] Figure 11. Stimulation of human colonic epithelial cells by compounds / products according to the invention. Human colonic epithelial cells (HCT 1 16) that were either wild type (WT) or deficient in TIFA protein expression (knockout, KO) were stimulated for 20 minutes in permeabilization buffer (5 pg/mL digitonin) in the presence of water or 10 pg/mL, 30 pg/mL, or 50 pg/mL of indicated synthetic compounds according to the invention. Treatment was removed, cells were washed, and cells were incubated for 6 hours in complete media before IL-8 levels in culture supernatants was measured by ELISA. The results are the mean ± standard error of the mean of three technical replicates. JS-10: 7-0-(Aminoethyl)-D-glycero-3-D-manno-heptopyranose phosphate; JS-11 : 7-0- (Aminoethyl)-D-glycero-D-manno-heptopyranose; JS-12: D-glycero-D-manno-heptose; Js-13: pH- Rhodo-7-0-(aminoethyl)-D-glycero-3-D-manno-heptopyranose phosphate. [0036] Figure 12. Effects of compounds / products according to the invention on HEK 293T cells encoding an NF-KB-driven luciferase reporter gene. HEK 293T cells were transfected with a plasmid encoding an NF-KB-driven luciferase reporter. After 24 hours, cells were stimulated for 20 minutes in permeabilization buffer (5 pg/mL digitonin) in the presence of water or 10 pg/mL, 30 pg/mL, or 50 pg/mL of indicated synthetic compounds according to the invention. Treatment was removed; cells were washed and incubated for 6 hours in complete medium. A luciferase assay was then performed. JS-12a control: D-glycero-D-manno heptose. The results are the means ± standard error of the mean of three technical replicates. JS-10: 7-0-(Aminoethyl)-D-glycero-3-D-manno-heptopyranose phosphate; JS-11 : 7-0-(Aminoethyl)-D-glycero-D-manno-heptopyranose; JS-12: D-glycero-D- manno-heptose; Js-13: pH-Rhodo-7-0-(aminoethyl)-D-glycero- -D-manno-heptopyranose phosphate.
[0037] Figure 13. Stimulation of human colonic epithelial cells by compounds / products according to the invention. Human colonic epithelial cells (HCT 1 16) that were either wild type (WT) or deficient in TIFA protein expression (knockout, TIFA KO) were stimulated for 20 minutes in permeabilization buffer (5 pg/mL digitonin) in the presence of water, 20 pg/mL of the Nodi agonist C12-E-DAP (39.8 pM, which induces inflammation in a TIFA-independent manner) or 30 pM or 150 pM of synthetic compound according to the invention. Treatment was removed, cells were washed, and cells were incubated for 6 hours in complete media before IL-8 levels in culture supernatants were measured by ELISA. The results are the mean ± standard error of the mean of two (150 pM treatment) or three (all other treatments) independent experiments. # = none detected, JS-10: 7-0- (Aminoethyl)-d-glycero- -d-manno-heptopyranose phosphate; JS-14: Biotin-7-0-(aminoethyl)-d- glycero- -d-manno-heptopyranose phosphate, Nodi agonist: C12-iE-DAP (20pg/ml, 39.8mM).
[0038] Figure 14. Effects of compounds / products according to the invention on HEK 293T cells encoding an NF-KB-driven luciferase reporter gene. HEK 293T cells were transfected with a plasmid encoding an NF-KB-driven luciferase reporter. After 24 hours, cells were stimulated for 20 minutes in permeabilization buffer (5 pg/mL digitonin) in the presence of water, or either 30 pM or 150 pM of indicated synthetic compounds according to the invention. Treatment was removed; cells were washed and incubated for 6 hours in complete medium. A luciferase assay was then performed. The results are the mean and standard error of the mean of three technical replicates and are representative of three independent experiments. JS-7: JS-7:D-glycero-p-D-manno-heptose-phosphate; JS-10: 7-0-(Aminoethyl)-D-glycero- -D-manno-heptopyranose phosphate; JS-14: Biotin-7-O-
(aminoethyl)-D-glycero- -D-manno-heptopyranose phosphate. [0039] Figure 15. Stimulation of human macrophages by compounds / products according to the invention. Human macrophage cells (THP-1) were stimulated for 20 minutes in permeabilization buffer (5 pg/mL digitonin) in the presence of water, 39.8 pM of the Nodi agonist C12-E-DAP (which stimulates in a TIFA-independent manner), or either 30 pM or 150 pM of synthetic compound according to the invention. T reatment was removed, cells were washed, and cells were incubated for 6 hours in complete media before the IL-8 levels in culture supernatants were measured by ELISA. The results are the mean and standard error of the mean of three technical replicates. Nodi agonist: C12- iE-DAP (20pg/ml, 39.8pM); JS-7: JS-7: D-glycero- -D-manno-heptose-phosphate; JS-10: 7-0- (Aminoethyl)-D-glycero- -D-manno-heptopyranose phosphate; JS-14: Biotin-7-0-(aminoethyl)-D- glycero- -D-manno-heptopyranose phosphate.
[0040] Figure 16. Stimulation of human colonic epithelial cells by compounds / products according to the invention. Human colonic epithelial cells (HCT 1 16) that were either wild type (WT) or deficient in TIFA protein expression (knockout, TIFA KO) were stimulated for 20 minutes in permeabilization buffer (5 pg/mL digitonin) in the presence of water, 20 pg/mL of the Nodi agonist C12-E-DAP (39.8 pM, which induces inflammation in a TIFA-independent manner) or 30 pM or 150 pM of synthetic compound according to the invention. Treatment was removed, cells were washed, and cells were incubated for 6 hours in complete media before IL-8 levels in culture supernatants were measured by ELISA. The results are the mean ± standard error of three technical replicates. JS-24a: 7-0-([Biotin]-dPEG4-amidoethyl)-D-Glycero-p-D-manno-heptopyranosyl phosphate; JS- 25a: 7-0-([Fluorescein]-amidoethyl)-D-Glycero-p-D-manno-heptopyranosyl phosphate, Nodi agonist: C12-iE-DAP (20pg/ml, 39.8mM).
[0041] Figure 17. Effects of compounds / products according to the invention on HEK 293T cells encoding an NF-KB-driven luciferase reporter gene. HEK 293T cells were transfected with a plasmid encoding an NF-KB-driven luciferase reporter. After 24 hours, cells were stimulated for 20 minutes in permeabilization buffer (5 pg/mL digitonin) in the presence of water, TNF-a (80ng/mL) or either 30 pM or 150 pM of indicated synthetic compounds according to the invention. Treatment was removed; cells were washed and incubated for 5 hours in complete medium. A luciferase assay was then performed. The results are the mean ± standard error of three technical replicates. JS-24a: 7-0-([Biotin]- dPEG4-amidoethyl)-D-Glycero-p-D-manno-heptopyranosyl phosphate; JS-25a: 7-0-
([Fluorescein]-amidoethyl)-D-Glycero-p-D-manno-heptopyranosyl phosphate. Abbreviations
[0042] CRM: cross-reacting material (a genetic mutant of diphtheria toxin); HBP: heptose-1 , 7-biphosphate or D-g/ycero-D-manno-heptopyranose-1 ,7-p-phosphate; HMP- b: D-g/ycero-D-manno-heptopyranose-i p-phosphate (also named heptose-1- monophosphate or JS7); PAMP: pathogen associated molecular pattern; GFP: green fluorescent protein; Sup: supernatant; WT: wild type; TIFA: tumor necrosis factor receptor-associated factor (TRAF)-interacting protein with forkhead-associated domain; TIFA KO: TIFA protein expression knockout.
Definitions
[0043] In order to provide a clear and consistent understanding of the terms used in the present specification, a number of definitions are provided below. Moreover, unless defined otherwise, all technical and scientific terms as used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains.
[0044] The use of the word “a” or“an” when used in conjunction with the term “comprising” in the claims and/orthe specification may mean“one”, but it is also consistent with the meaning of“one or more”,“at least one”, and“one or more than one”. Similarly, the word“another” may mean at least a second or more.
[0045] As used herein, the wording "and/or" is intended to represent an inclusive-or. That is, "X and/or Y" is intended to mean X or Y or both, for example. As a further example, "X, Y, and/or Z" is intended to mean X or Y or Z or any combination thereof.
[0046] As used in this specification and claim(s), the words“comprising” (and any form of comprising, such as“comprise” and“comprises”), “having” (and any form of having, such as“have” and“has”),“including” (and any form of including, such as“include” and “includes”) or“containing” (and any form of containing, such as “contain” and “contains”), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.
[0047] As used in this specification and claim(s), the terms“Ci-6 » (or more generally “Cx-y”) means a chain containing from 1 to 6 carbon atoms (or more generally x to y carbon atoms), being saturated or unsaturated, linear or branched, as permitted by the laws of organic chemistry, well understood by the person skilled in the art. [0048] As used herein, the term“phosphorylated heptose compound” refers to a monosaccharide with seven carbon atoms, wherein at least one hydroxyl group is replaced by a group comprising a phosphorus atom. For example, the term “mono- phosphorylated heptose compound” refers a monosaccharide with seven carbon atoms, wherein one hydroxyl group is replaced by a group comprising a phosphorus atom. The term also refers to a derivative or an analogue of such compound.
[0049] As used herein, the term“label”,“marker” or“tag” refer to any molecule used in such a way as to attach a functional or diagnostic or other moiety to the molecule, such as but not limited to fluorescent tags, affinity tags, transfer factor, plasmids or lipids, in such a way that the bioactivity can be for example but not limited to followed, targeted, directed or modified or otherwise guided, analyzed or assessed.
[0050] As used herein, the term “modulate” in connection with an immune or inflammatory response refers to a qualitative or quantitative alteration in the immune or inflammatory response in a subject.
[0051] As used herein, the term “vaccine” or “vaccine composition” refers to a pharmaceutical composition containing an immunogen. The composition may be used for modulating an immune response in a subject. The term also refers to subunit vaccines, i.e. , vaccine compositions containing immunogens which are separate and discrete from a whole organism with which the immunogen is associated in nature.
[0052] As used herein, the term “effective amount” refers to the amount of a compound medicinal molecule or reaction product sufficient to cure, alleviate or partially arrest the clinical manifestations of a given disease and its complications in a therapeutic intervention comprising the administration of said compound or reaction product. An effective amount for each purpose will depend on the severity of the disease or injury as well as the weight and general state of the subject.
[0053] As used herein, the term“functional molecule” refers to a molecule that is administrable to a mammal for modulating or labeling a least one physiological parameter, marker or target in a cell, organ, biological system, organism or mammal.
[0054] In general, the methods of the present disclosure may be used to therapeutically or prophylactically treat any subjects for which increased activation of the immune system or an altered immune response would be beneficial. This includes, but is not restricted to a subject suffering from a condition which deleteriously affects the immune system, including any subjects at a heightened risk of infection or actually infected, for example due to surgery or imminent surgery, injury, illness, radiation or chemotherapy, and any subjects suffering from autoimmune diseases, inflammatory disorders, cancers, and diseases which cause the normal metabolic immune response to be compromised, such as HIV (AIDS).
[0055] As used herein the terms “prevention” and “preventing” mean the management and care of a subject for the purpose of preventing the occurrence of a condition, such as a disease or disorder. The composition may be used for inducing or enhancing an immune response against a potential pathogen in a subject for blocking infection, delaying onset or decreasing transmission. The subject to be treated is preferably a mammal, in particular a human being.
[0056] As used herein the terms“treatment” and“treating” mean the management and care of a subject for the purpose of combating a condition, such as a disease or disorder. The term is intended to include the full spectrum of treatments for a given condition from which the patient is suffering, such administration of the active compounds to alleviate the symptoms or complications, to delay the progression of the condition, and/or to cure or eliminate the condition. The subject to be treated is preferably a mammal, in particular a human being.
[0057] As used herein, the term “subject” is understood as being any mammal including a human being treated with a compound of the invention.
[0058] Before the present invention is further described, it is to be understood that the invention is not limited to the particular embodiments described below, as variations of these embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments and is not intended to be limiting. Instead, the scope of the present invention will be established by the appended claims.
DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS
[0059] The technology is directed to b-D-heptose-l -phosphate (HMP)-linker derivatives and molecule conjugates of b-D-heptose-l -phosphate (HMP) for modulating an immune response in a subject. Observation of biological activity of the HMP molecule was not at all expected based on prior art. Indeed, previous studies had indicated that loss of the 7-phosphate from the 1 ,7-biphosphate molecule eliminated activity. This observation of activity of the 1-phosphate (HMP) linker provides a unique opportunity to fully understand the scope of the immunomodulatory possibilities and provide a reproducible methodology that could provide milligram to gram quantities of a potentially valuable biological molecule. The proof of activity of the HMP creates two major advantages.
[0060] Firstly, the synthesis of the HMP is significantly simpler and likely more commercially realisable than that of the HBP. Secondly, this creates the potential opportunity to modify the 7-position of the HMP to link this immunomodulator to a huge variety of functional molecules whose processing and activity may be controlled and directed by conjugation to this PAMP-like molecule.
[0061] Experiments involved replacing the terminal OH group of the glycerol moiety in HMP with another chemical entity to introduce a linkage moiety, were carried out to facilitate the production of novel immunomodulatory compounds with improved protection and efficiency.
[0062] The inventors have identified phosphorylated heptose-derivatives useful for conjugating with a functional molecule and modulating their immunomodulatory activities thereof. These conjugates may be useful in modulating (i.e. enhancing or decreasing) an immune response in a subject or labeling the molecule in such a way that it may be followed, targeted, directed or modified or otherwise guided.
HMP-Linker
[0063] In accordance with a particular embodiment of the present invention, there is provided an HMP-linker compound of formula (I) or (la):
(la), wherein R is a linkage moiety for conjugating with a functional molecule. Particularly, the resulting HMP-conjugate is capable of modulating an immune response in a subject.
[0064] In accordance with a particular embodiment, R is selected from the group consisting of: -C(0)0H, -C(0)H, -Co-6alkyl-C(0)-Ci-6 alkyl, -Co-6alkyl-N3, -Ci-6 alkyl, -C0- 6alkyl -0-Ci-6 alkyl, -Co-6alkyl-aryl, -Co-6alkyl-0-aryl, -Co-6alkyl-C2-6-allyl groups, and -C0- 6alkyl-0-C2-6-allyl, wherein said -Co-6alkyl-C(0)-Ci-6 alkyl, -Ci-6 alkyl, C0-6alkyl-O-Ci-6 alkoxy, -aryl, C0- ealkyl-O-aryl, Co 6alkyl-C2 e-allyl groups, and Co-6alkyl-0-C2-6-allyl is optionally substituted with a: amino, acyloxy, alkoxy, carboxyl, carbalkoxyl, hydroxy, trifluoromethyl, cyano, nitro, acyl, or a halo group;
or R is selected from the group consisting of: -Ci-6 alkyl-0P03C(0)-Ci-6 alkyl, -Ci-6 alkyl- OPO3C1-6 alkyl, -C1-6 alkyl-OP03-Ci-6-0-alkoxy, -C1-6 alkyl-0P03-aryl, -C1-6 alkyl-OPC>3-C2- 6-allyl,
wherein said -Ci-6 alkyl-0P03C(0)-Ci-6 alkyl, -Ci-6 alkyl-0P03Ci-6 alkyl, -Ci-6 alkyl- OP03-Ci-6-0-alkoxy, -Ci-6 alkyl-0P03-aryl, -Ci-6 alkyl-0P03-C2-6-allyl group is optionally substituted with a: amino, acyloxy, alkoxy, carboxyl, carbalkoxyl, hydroxy, trifluoromethyl, cyano, nitro, acyl, or a halo group;
or a salt thereof.
[0065] In accordance with a particular embodiment, R is selected from the group consisting of: -C(0)0H, -C(0)H, -C(0)-Ci-6 alkyl, - N3, -C1-6 alkyl, -O-C1-6 alkyl, -aryl, -O- aryl, -C2-6-allyl groups, and -0-C2-6-allyl, wherein said -C(0)-Ci-6 alkyl, -Ci-6 alkyl, -0-Ci-6 alkoxy, -aryl, -O-aryl, -C2-6-allyl groups, and -0-C2-6-allyl is optionally substituted with : amino, acyloxy, alkoxy, carboxyl, carbalkoxyl, hydroxy, trifluoromethyl, cyano, nitro, acyl, or a halo group;
or R is selected from the group consisting of: CH20P03C(0)-CI-6 alkyl, CH20P03Ci-6 alkyl, CH20P03-CI-6-0 alkoxy, CH20P03-aryl, CH20P03-C2-6-allyl, wherein said CH20P03C(0)-CI-6 alkyl, CH20P03Ci-6 alkyl, CH20P03-Ci-6-0 alkoxy, CH20P03-aryl, CH20P03-C2-6-allyl group is optionally substituted with a: amino, acyloxy, alkoxy, carboxyl, carbalkoxyl, hydroxy, trifluoromethyl, cyano, nitro, acyl, or a halo group; or a salt thereof.
[0066] In accordance with a particular embodiment, R is selected from the group consisting of: -Ci-6alkyl, -C(0)0H, -C(0)H, -C(0)-Ci-6 alkyl, -Co-6alkyl-0-Ci-6 alkyl, -C0- 6a I ky l-C 2-6- a I ly I groups, and -0-C2-6-allyl, wherein each of said -Ci_6alkyl, -C(0)-Ci-6 alkyl, -Co-6alkyl-0-Ci-6 alkyl, _-Co-6alkyl-C2-6-allyl, and -Co-6alkyl-0-C2-6-allyl group is optionally substituted with a : amino, alkoxy, hydroxy, carboxy or nitro group;
or R is selected from the group consisting of: CH20P03C(0)-CI-6 alkyl, CH20P03Ci-6 alkyl, CH20P03-CI-6-0 alkoxy, CH20P03-aryl, CH20P03-C2-6-allyl, wherein each of said CH20P03C(0)-CI-6 alkyl, CH20P03Ci-6 alkyl, CH20P03-Ci-6-0 alkoxy, CH20P03-aryl, CH20P03-C2-6-allyl group is optionally substituted with a: amino, alkoxy, hydroxy, or nitro group; or a salt thereof. [0067] In accordance with a particular embodiment of the present invention, there is provided an HMP-linker compound of formula (la):
wherein R is a linkage moiety for conjugating with a functional molecule. Particularly, the resulting HMP-conjugate is capable of modulating an immune response in a subject.
[0068] In accordance with a particular embodiment, R is selected from the group consisting of: -C(0)-Ci-6 alkyl, - N3, -C1-6 alkyl, -O-C1-6 alkyl, -aryl, -O-aryl, -C2-6-allyl groups, and -0-C2-6-allyl, wherein each of said -C(0)-Ci-6 alkyl, -Ci-6 alkyl, -0-Ci-6 alkoxy, -aryl, - O-aryl, -C2-6-allyl, and -0-C2-6-allyl groups is optionally substituted with a : amino, alkoxy, hydroxy, or nitro group; or R is selected from the group consisting of: CH20P03C(0)-CI-6 alkyl, CH20P03CI-6 alkyl, CH20P03-CI-6-0 alkoxy, CH20P03-aryl, CH20P03-C2-6-allyl, wherein each of said CH20P03C(0)-CI-6 alkyl, CH20P03CI-6 alkyl, CH20P03-CI-6-0 alkoxy, CH20P03-aryl, CH20P03-C2-6-allyl group is optionally substituted with a: amino, alkoxy, hydroxy, or nitro group. [0069] In accordance with a particular embodiment, the HMP-linker of formula (I) or
(la) is substituted with an“R” group that may be selected from the group consisting of: - C(0)0H, -C(0)H, -C(0)Ci-6 alkyl, -CH2N3, -CH2OCI-6 alkyl, -CH2Oaryl, and -CH2OC2.6- allyl groups, wherein said -C(0)Ci-6 alkyl, -CH2OCI-6 alkyl, -CH2Oaryl, and -CH2OC2.6- allyl group is optionally substituted with a : amino, acyloxy, alkoxy, carboxyl, carbalkoxyl, hydroxy, trifluoromethyl, cyano, nitro, acyl, or a halo group; or a salt thereof. More particularly, the group R is: CH2O-CH2CH2-NH2; -C(0)OH, -C(0)H, -C(0)NH2 or -CH2N3 or CH2NH2.
[0070] Additionally, it is possible to modify HMP via a phosphate residue, wherein R is CH20P03C(0)-CI-6 alkyl, CH20P03Ci-6 alkyl, CH20P03-Ci-6-0 alkoxy, CH20P03-aryl, CH2OPC>3-C2-6-allyl. wherein said CH20PC>3C(0)-CI-6 alkyl, CH2OPC>3CI-6 alkyl, CH20P03-CI-6-0 alkoxy, CH20P03-aryl, CH20P03-C2-6-allyl groups, is optionally substituted with a: amino, acyloxy, alkoxy, carboxyl, carbalkoxyl (alkoxycarbonyl), hydroxy, trifluoromethyl, cyano, nitro, acyl, or a halo group; or a salt thereof. [0071] More particularly, R is selected from the group consisting of: Ci-6alkyl-NH2; - Co-6 alkyl-C-O-Co-6 alkyl-NH2, -C(0)0H, -C(0)H, -C0-6 alkyl-C(O)C0-6 alkyl-NH2, C0-6alkyl- N3. Most particularly, R is selected from the group consisting of:CH2-NH2; -C(0)0H, - C(0)H, -C(0)NH2 and - N3. [0072] More particularly, R is selected from the group consisting of: CH2-NH2; -
C(0)0H, -C(0)H, -C(0)NH2 and - N3.
[0073] According to a particular embodiment, the HMP-linker may be chosen from:
Conjugates
[0074] These conjugate-compounds (or conjugates) can now be targeted to the unique arm of the immune system that processes HMP / HBP, thus potentially enhancing the immunological impact of the conjugated functional molecule.
[0075] In accordance with a particular aspect of the invention, there is provided a compound comprising a functional molecule conjugated to a D-glycero-D-manno- heptopyranose I b-phosphate (heptopyranose-1 -monophosphate; HMP) moiety. In a particular embodiment, the functional molecule is conjugated at position-7 of HMP, or -2 of HMP. Particularly, the functional molecule is conjugated at position-7 of HMP. [0076] In accordance with a particular embodiment, the invention provides a conjugate compound of formula (II) as defined herein, for use in targeting and/or modulating an immune response in a subject.
[0077] According to a particular embodiment, the conjugate-compound is defined by formula (I la):
(lla), wherein X is a conjugated functional molecule.
Molecules for conjugating to HMP-linker
[0078] In accordance with a particular embodiment, the HMP-linker is conjugated to a functional molecule for modulating a host’s immune response to this functional molecule. Particularly, the functional molecule is a protein (e.g. carrier proteins such as: CRM, protein D, or TT), polypeptide (e.g. Ova-specific peptide: CGGSIINFEKLTEWTSSNVMEER (SEQ ID No.1)), an antibody, a lipid (e.g. glycerolipid), a fatty acid (e.g. stearic acid or palmitic acid), a carbohydrate, a drug, a fluorescent tag, an affinity tag or a transfer factor molecule. Particularly, the functional molecule is an antigen, a biological response modifier (BRM, such as a hormone, immunogen, antibody, etc.) or a marker.
[0079] Alternatively, the functional molecule is a drug such as, for example, an anti- HIV drug including, but not limited to: Nucleoside/Nucleotide reverse transcriptase inhibitors (NRTI), such as for example, abacavir (ziagen), efavirenz/emtriacitabine/tenofovir disoproxil fumarate (atripla), lamivudine/zidovudine (combivir), emtriacitabine/rilpivirine/tenofovir disoproxil fumarate (complera), emtricitabine (emtriva), lamivudine (Epivir), abacavir/lamivudine (Epzicom), zidovudine (Retrovir), abacavir/lamivudine/zidovudine (Trizivir), emtricitabine/tenofovire disoproxil fumarate (Truvada), didanosine (Videx), didanosine extended release (Videx EC), tenofovir disoproxil fumarate (Viread), stavudine (Zerit); Multiclass combination drugs : such as efavirenz/emtricitabine/tenofovir disoproxil fumarate (Atripla), emtricitabine / rilpivirine / tenofovir disoproxil fumarate (Complera), elvitegravir / cobicistat/emtricitabine / tenofovir disoproxil fumarate (Stribild), abacavir / dolutegravir/lamivudine (Triumeq); Nonnucleoside reverse transcriptase inhibitors (NNRTIs), such as, but not limited to: rilpivirine (Edurant), etravirine (Intelence), delavirdine mesylate (Rescriptor), efavirenz (Sustiva), nevirapine (Viramune), nevirapine extended-release (Viramune XR); Protease inhibitors(PI) suhc as, but not limited to: tipranavir (Aptivus), indinavir (Crixivan), atazanavir/cobicistat (Evotaz), saquinavir (Invirase), lopinavir/ritonavir (Kaletra), fosamprenavir (Lexiva), ritonavir (Norvir), darunavir/cobicistat (Prezcobix), darunavir (Prezista), atazanavir (Reyataz), nelfinavir (Viracept); Entry inhibitors (including fusion inhibitors) such as, but not limited to: enfuvirtide (Fuzeon); Integrase inhibitors, such as, but not limited to: raltegravir (Isentress), dolutegravir (Tivicay), elvitegravir (Vitekta); Chemokine co-receptor antagonists (CCR5 antagonists), such as, for example: maraviroc (Selzentry); Cytochrome P4503A (CYP3A) inhibitors, such as, but not limited to: cobicistat (Tybost).
[0080] Some anti-HCV drugs, such as, for example: protease inhibitors (PI) approved for hepatitis C only are sometimes prescribed by doctors for HIV: simeprevir (Olysio), boceprevir (Victrelis), ombitasvir/paritaprevir/ritonavir/dasabuvir (Viekira Pak).
[0081] The functional molecule that could be linked to HMP may include anti-cancer drugs, such as, but not limited to: Abiraterone, Alemtuzumab, Anastrozole, Aprepitant, Arsenic trioxide, Atezolizumab, Azacitidine, Bevacizumab, Bleomycin, Bortezomib, Cabazitaxel, Capecitabine, Carboplatin, Cetuximab, Chemotherapy drug combinations, Cisplatin, Crizotinib, Cyclophosphamide, Cytarabine, Denosumab, Docetaxel, Doxorubicin, Eribulin, Erlotinib, Etoposide, Everolimus, Exemestane, Filgrastim, Fluorouracil, Fulvestrant, Gemcitabine, HPV Vaccine, Imatinib, Imiquimod, Ipilimumab, Ixabepilone, Lapatinib, Lenalidomide, Letrozole, Leuprolide, Mesna, Methotrexate, Nivolumab, Oxaliplatin, Paclitaxel, Palonosetron, Pembrolizumab, Pemetrexed, Prednisone, Radium-223, Rituximab, Sipuleucel-T, Sorafenib, Sunitinib, Talc Intrapleural, Tamoxifen, Temozolomide, Temsirolimus, Thalidomide, Trastuzumab, Vinorelbine, or Zoledronic acid.
[0082] Alternatively, the functional molecule is an antigen, more particularly a molecule that targets a cell differentiation antigen, such as a-specific binding partner for targeting the HMP moiety to a particular type of cells, be it cancer cells, immune cells, or cells of any particular organ or location in the body.
Antigen for targeting specific cells
[0083] Particularly, when the conjugated functional molecule is a molecule that targets a cell differentiation antigen, such as a specific binding partner, this molecule may be used as a“magic bullet” to target the immunomodulatory HMP moiety to a particular type of cells, such as, for example, HIV reservoir cells.
Cleavage of HMP moiety
[0084] Alternatively, once the conjugate has reached its desired cell-type, the HMP moiety may be cleaved from the conjugate to release the HMP moiety to the targeted cell type. For this to be achieved, the person skilled in the art will adapt the linkage-moiety to be used for cleavage to be carried out once the conjugate has reached the appropriate cell type and release the HMP immunomodulatory moiety.
[0085] Particularly, the cleavable linker can have cleavable unit that is a photocleavable, enzyme-cleavable or chemically- cleavable unit. For example, the cleavable linker can have a cleavable unit such as a disulfide (chemically cleavable), nitrobenzo (a photocleavable unit), or amine, amide or ester (enzyme-sensitive cleavable units). More particularly, the cleavable linker can be of the formula: X - s - S - Z, or, more
particularly: Immunogen
[0086] In accordance with a particular embodiment of the invention, the functional molecule conjugated to HMP is an immunogen, particularly an immunogen expressed by, or derived from, an infectious agent, such as for example an infectious bacterial, viral or parasitic pathogens, including Gram-negative bacterial pathogens belonging to the genus Neisseria (including Neisseria meningitidis, Neisseria gonorrohoeae), Escherichia (including Escherichia coli), Klebsiella (including Klebsiella pneumoniae), Salmonella (including Salmonella typhimurium), Shigella (including Shigella dysenteriae, Shigella flexneri, Shigella sonner), Vibrio (including Vibrio cholerae), Helicobacter (including Helicobacter pylori), Pseudomonas (including Pseudomonas aeruginosa), Burkholderia (including Burkholderia multivorans), Haemophilus (including Haemophilus influenzae), Moraxella (including Moraxella catarrhalis), Bordetella (including Bordetella pertussis), Francisella (including Francisella tularensis), Pasteurella (including Pastourelle multocida), Legionella (including Legionella pneumophila), Borrelia (including Borrelia burgdorferi), Campylobacter (including Campylobacter jejuni), Yersinia (including Yersinia pestis and Yersinia enterocolitica), Rickettsie (including Rickettsia rickettsii), Treponema (including Treponema pallidum), Chlamydia (including Chlamydia trachomatis, Chlamydia pneumoniae) and Brucella spp., and including Gram positive bacterial pathogens belonging to the genus Staphylococcus (including Staphylococcus aureus), Streptococcus (including Streptococcus pneumoniae, Streptococcus pyogenes), Listeria (including Listeria monocytogenes), Corynebacterium (including Corynebacterium diphtheriae), Enterococcus (including Enterococcus faecalis), Clostridium spp., and Mycobacterium (including Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium avium).
[0087] Immunogens may also be derived from pathogenic viruses including Adenoviridae (including Adenovirus), Herpesviridae (including Epstein-Barr virus, Herpes Simplex Viruses, Cytomegalovirus, or Varicella Zoster virus), Papillomviridae, Poxviridae (including Papillomavirus), Hepadnaviridae (including Hepatitis B virus), Parvoviridae, Astroviridae, Caliciviridae, Picornaviridae (including Coxsackievirus, Hepatitis A virus, Poliovirus), Coronaviridae, Flaviviridae (including Hepatitis C virus, Dengue virus), Togaviridae (including Rubella virus), Hepeviridae, Retroviridae (including HIV), Orthomyxoviridae (including influenza virus, Arenaviridae, Bunyaviridae, Filoviridae, Paramyxoviridae (including Measles virus, Mumps virus, Parainfluenza virus, Respiratory Syncytial virus), Rhabdoviridae (including Rabies virus) or Reoviridae.
[0088] Immunogens may also be derived from pathogenic fungal infections including those caused by Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis, or Coccidioides. Vaccines may also target parasitic pathogens including Leishmania, Plasmodium, Toxoplasma, Trypanosoma and Schistosoma.
[0089] The immunogen may be derived from a protein or other molecules expressed on or within the subject's own cells, such as a tumor-specific immunogen or cancer- specific immunogen, to stimulate an immune response against the pathogenic cells or tissues. In one embodiment, the HBP may be introduced directly into a tumor to increase the immune response against the tumor.
[0090] According to a particular embodiment, the HMP-immunogen conjugate of the invention can be administered as part of a vaccine formulation.
Markers or labels
[0091] In accordance with a particular embodiment, the linker of the invention is conjugated to a marker, such as, for example, a label or a tag. Particularly, the marker or tag is chosen from fluorescent and / or enzymatic tags, such as but not limited to: biotin, rhodamine, fluorescein, CF tags, EvoBlue, rhodopsin, GFP, fluorescent red, fluorescent orange biotin or other labels such as, for example:
(see www.sigmaaldrich.com/life-science/biochemicals/biochemical-products. html?TablePage=16187968)
[0092] Most particularly, the compound of formula (I) is chosen from:
Synthesis
[0093] In accordance with an alternative aspect of the invention, there is provided a method for the synthesis of a compound of formula (I) comprising the steps of:
[0094] Compound 7 was synthesized as described in Sauvageau et al., Carbohydr. Res., 2017, 450, 38-43 [18]. In short:
a) the hydroxy of compound 7 was protected with a cyanoethyl moiety before b) hydroxylation of the double bond;
c) then the nitrile was reduced to the amine and was protected with a P1 protecting group;
d) cleavage of the acetonide and P1 group followed by acetylation gave acetylated compound 21 ;
e) after deprotection of the anomeric acetyl; and
f) phosphorylation;
g) the compound was globally deprotected to form JS10a;
j) conjugation to molecule X activated ester gave (lla) corresponding to a specific compound of formula (II).
[0095] A variety of linkers are synthesized to understand how the nature of the linker may impact the biological activity. With this knowledge, a vast array of functional molecules are appropriately attached to an HMP-linker to examine several aspects of this PAMP’s interaction with the immune system and to augment and specifically target the linked entities processing within the immune system.
[0096] These can include but are not limited to, the identification of the receptor for HMP, an understanding of the processing of HMP, developing antibodies to HMP to follow the trafficking of the molecule, or targeting HMP to certain cells within the immune system by linking to a fluorescent moiety for example.
[0097] In accordance with a further aspect of the invention, there is provided a method for the preparation of a therapeutic conjugate having modulated
immunomodulatory activity, the method comprising the step of: conjugating a functional molecule with a compound of formula (I) or (la) as defined herein.
[0098] The conjugates of the present invention can be synthesized via the synthesis of HMP that is significantly simpler and likely more commercially realisable than that of HBP.
Compound for use and uses
[0099] In accordance with a further aspect of the invention, there is provided a conjugate compound of formula (II) or (lla) as defined herein, for use in targeting and/or modulating an immune response in a subject. Particularly, the ability to target the modulation of the immune response of the subject is useful for preventing, treating, ameliorating, or inhibiting an injury, disease, disorder or condition wherein the modulation of the immune response is beneficial.
[00100] According to a particular embodiment, the conjugate compound for use is carried out in the context of a vaccine for the treatment or prevention of disease.
[00101] In accordance with a further aspect of the invention, there is provided a use of an effective amount of a conjugate, also called a compound of formula (II) or (lla) as defined herein, for targeting and/or modulating an immune response in a subject. Particularly, the modulation of the immune response of the subject is useful for preventing, treating, ameliorating, or inhibiting an injury, disease, disorder or condition wherein the modulation of the immune response is beneficial.
[00102] According to a particular embodiment, the use of the compound is carried out in the context of a vaccine for the treatment or prevention of disease. [00103] Alternatively, the use of the compound of formula (II) or (lla as defined herein, is carried out for the manufacture of a medicament for modulating an immune response in a subject.
[00104] Particularly, in the context of the use of the conjugate of the invention, the immune response of the subject is enhanced. In this embodiment, the molecule conjugated to the HMP-linker is an antigen or an immunogen, more particularly chosen from cell-differentiation antigen, or antigens from cancer cells, bacteria, viruses or parasites.
[00105] More particularly, the immunogen used for embodying the invention is from Gram-negative bacteria, most particularly chosen from: Neisseria, Escherichia, Klebsiella, Salmonella, Shigella, Vibrio, Helicobacter, Pseudomonas, Burkholderia, Haemophilus, Moraxella, Bordetella, Francisella, Pasteurella, Borrelia, Campylobacter, Yersinia, Rickettsia, Treponema, Chlamydia and Brucella.
[00106] Still more particularly, the immunogen useful for the invention is from Grampositive bacteria, most particularly chosen from: Staphylococcus, Streptococcus, Listeria, Corynebacterium, Enterococcus, Clostridium and Mycobacterium.
[00107] Alternatively, the immunogen used for embodying the present invention is derived, or comes, from a virus, more particularly chosen from: Adenoviridae, Herpesviridae, Papillomviridae, Poxviridae, Hepadnaviridae, Parvoviridae, Astroviridae, Caliciviridae, Picornaviridae, Coronaviridae, Flaviviridae, Togaviridae, Hepeviridae, Retroviridae, Orthomyxoviridae, Arenaviridae, Bunyaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, Reoviridae and Human Immunodeficiency (HIV) viruses.
[00108] Still, according to an alternative embodiment, the immunogen is from a parasite, more particularly chosen from: Leishmania, Plasmodium, Toxoplasma, Trypanosoma and Schistosoma.
[00109] Still, alternatively, the immunogen may originate from a cancer-specific antigen, more particularly some or all of the cancer-specific antigens listed in Table 1 : Table 1 (cancer-specific antigen useful for conjugating with HMP-linker
Category Example Antigen Cancer Histology
Oncofetal CEA Colorectal carcinoma
Immature laminin receptor RCC
TAG-72 Prostate carcinoma
Oncoviral HPV E6, E7 Cervical carcinoma
Overexpressed/accumulated BING-4 Melanoma
Calcium-activated chloride channel 2 Lung carcinoma
Cyclin-B j Multi
9D7 RCC
Ep-CAM Breast carcinoma
EphA3 Multi
Her2/neu Multi
Telomerase Multi
Mesothelin Ductal pancreatic carcinoma
SAP-1 Colorectal carcinoma
Survivin Multi
Cancer-Testis BAGE family Multi
CAGE family Multi
GAGE family Multi
MAGE family Multi
SAGE family Multi
XAGE family Multi
CT9, CT10 Multi
NY-ESO-l/LAGE-1 Multi
PRAME Multi
SSX-2 Melanoma, Multi Lineage Restricted Melan-A/MART-1 Melanoma
Gpl00/pmell7 Melanoma
Tyrosinase Melanoma
TRP-1/-2 Melanoma
P.polypeptide Melanoma
MC 1R Melanoma
Prostate-pecific antigen Prostate
Mutated b-catenin Melanoma, Prostate, HCC
BRCA1/2 Breast, ovarian carcinoma
CDK4 Multi
CML66 CML
Fibronectin Multi
MART-2 Melanoma p53 Multi
Ras Multi
TGF- RII Colorectal carcinoma
Posttranslationally altered MUC 1 Ductal carcinoma, RCC
Idiotypic Ig, TCR B, T leukemia, lymphoma, myeloma
BRCA = breast cancer antigen; CDK4 = cyclin-dependent kinase-4; CEA = carcino-embryonic antigen; CML66 = chronic myelogenous leukemia (antigen) 66; CT= cancer testis; HPV = human papilloma virus; Ep-CAM = epithelial cell adhesion molecule; Ig = immunoglobulin; MART-1/-2 = melanoma antigen recognized by T cells- 1/-2; MC 1R = melanocortin-1 -receptor; SAP- 1 = stomach cancer- associated protein tyrosine phosphatase- 1 ; TAG-72 = tumor antigen-72; TCR = T cell receptor; TGF-PRII = transforming growth factor-b receptor II; TRP = tyrosinase-related protein.
[00110] According to an alternative embodiment, there is provided a use of the conjugate compound of the invention, wherein the immune response of the subject is decreased, more particularly in the case where the immune response is provoked by an inflammatory response or an allergic reaction to a foreign element. In this case, the compound of the invention is conjugated with an immune-inhibitory molecule.
Method of treatment or prevention
[00111] In accordance with a further aspect, there is provided a method for modulating an immune response in a subject, comprising administering an effective amount of the compound of formula (II) or (I la) as defined herein to a subject suffering therefrom.
[00112] In accordance with a particular aspect, the modulation of immune response is necessary (or indicated or prescribed by a treating physician) for increasing an immune response to an infection caused by: bacteria, virus, or parasites, such as those defined herein above.
[00113] Alternatively, the modulation of immune response is necessary (or indicated or prescribed by a treating physician) for decreasing an immune response such as inflammation or an allergic reaction in a subject.
[00114] Particularly, the subject is a human.
[00115] In accordance with a particular embodiment, there is provided a method for the treatment of HIV comprising the steps of: administering an HMP-conjugated to an anti- HIV molecule, wherein the HMP moiety stimulates HIV latently-infected cells to start producing virus, and wherein the anti-HIV molecule kills the produced virus; whereby the HMP-anti-HIV conjugate eradicates the latent viral pool and treats the HIV infection.
[00116] In accordance with an alternative embodiment, there is provided a method for the treatment of HIV comprising the steps of: administering an HMP-conjugated to an HIV- reservoir cell-specific antigen binding partner to a subject, and treating said subject with an anti-HIV drug, wherein the conjugate-HMP moiety stimulates HIV latently-infected cells to start producing virus, and the anti-HIV drug kills the produced virus; whereby the HMP- anti-HIV conjugate eradicates the latent viral pool and treats the HIV infection.
[00117] Particularly, the anti-HIV molecule or drug is chosen from: Nucleoside/Nucleotide reverse transcriptase inhibitors (NNRTI), Multiclass inhibitors, Non-nucleoside reverse transcriptase inhibitors (NNRTIs), Protease inhibitors (PI), Entry inhibitors, Chemokine co-receptor antagonists (CCR5 antagonists), Cytochrome P4503A (CYP3A) inhibitors. More particularly, the anti-HIV drug is as defined herein above.
[00118] The present invention is illustrated in further details by the following nonlimiting examples.
EXAMPLES
[00119] The following examples are put forth to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
EXAMPLE 1 : CHEMICAL SYNTHESIS [00120] The molecules where data were obtained from are depicted in boxes in
Scheme 2 (also shown as Figure 1 B) (JS10a, JS11 a, JS13a, JS14a, JS24a, JS25a). JS12a refers to D-glycero-D-manno heptose control.
Scheme 2.
[Methoxybenzyl-7-0-cyanoethyl-(Z)-5,6-dioxy-2,3-0-isopropylidene-a-D-lyxo-hept-5- enofuran os id] uron ate (16).
[00121] Previously co-evaporated compound 7 (523 mg, 1 .6 mmol) was dissolved in acetonitrile (7 mL) and cooled down to 0°C. Sodium hydride (270 mg, 60% dispersion in mineral oil, 6.7 mmol) was added in portions and the mixture was stirred for 30 minutes. Bromoacetonitrile (500 pl_, 2.4 mmol) was added dropwise to the solution still kept at 0°C. The reaction mixture turned slowly dark brown and was shown to be completed after 5 hours (Rf = 0.5, ethyl acetate:hexane, 2:3, v:v). Methanol (5ml) was added slowly on ice and the reaction mixture was warmed up. The reaction mixture was dissolved in DCM, washed with water and brine and the organic layer was dried using magnesium sulfate. After concentration in vacuo, the compound was purifier using flash chromatography to give compound 16 in 88 % yield (525 mg, 1 .4 mmol). [a]D 25 = 46.7 (c = 0.01 , CH3CI3), 1H NMR (600 MHz, CDCI3): d= 7.25 (JCHCCH2, CHCOMe - 8.6 Hz, d, 2H, CHCCH?. PMB), 6.86 (^CHCOMe CHCCH 2 = 8.7 Hz, d, 2H, CHCOMe. PMB), 5.99 (m, 1 H, H5 ), 5.82 (m, 1 H, H6 ), 5.09 (s, 1 H, HT), 4.73 (JH4 , H3 = 3.6 Hz, JH4 ,H5 = 8.0 Hz, dd, 1 H, H4 ), 4.73 (JH3 ,H2 = 5.7 Hz, JH3',H4' = 3.9 Hz, dd, 1 H, H3 ), 4.63 (JH2 , H3 = 5.6 Hz, d, 1 H, H2 ), 4.62 (JCHA. CHB = 10.7 Hz, ABX, 1 H, CHA, PMB), 4.44 (JCHB CHA = 1 1 .5 Hz, ABX, 1 H, CHB, PMB), 4.24 (JH7A ,H6 = 1 .4 Hz, ABX, 1 H, H7A ), 4.23 (JH7B ,H6 = 1 .4 Hz, JH7B ,H6 = 6.5 Hz, ABX, 1 H, H7B ), 4.22 (s, 2H, CH2CN), 3.77 (3H, m, CH30, PMB), 1.44 (3H, s, CH3COCH2 ' ,OCH3), 1 .28 (3H, s, CH3COCH2 ' ,OCH3). 13C NMR (150MHz, CDCI3) d= 159.6 (COMe, PMB), 130.0 (CHCCH2 PMB), 129.7 (C, PMB and C5 ), 128.4 (C6 ), 1 16.2 (CN), 1 14.2 (CHCCH2 PMB), 1 12.8 (C2' ,3' Me02C), 105.4 (CT), 85.6 (C2 ), 81 .6 (C3 ), 75.9 (C4 ), 69.1 (CH2PMB), 67.2 (C7 ), 55.6 (OMe, PMB), 55.2 (CH2CN), 26.3 and 25.0 (CH3COCH2' ,OCH3 ). HRMS m/z Calcd for C2oH25N06Na [M+Na]+ 398.1580, found 398.1586.
Methoxybenzyl-7-0-(cyanoethyl)-2,3-0-isopropylidene-D-glycero-a-D-manno
heptofuranoside (17).
[00122] Alkene 16 (523 mg, 1.4 mmol) was stirred with NMMO (656 mI_, 2.8 mmol, 50% in water) for 30 minutes at RT in acetone:dioxane:water (12 mL, 1 :2: 1 , v:v:v). Then osmium tetraoxide (708 pL, 0.1 mmol, 4% in water) was added to the solution. The solution turned slowly yellow. After 5 hours, TLC monitoring showed that the reaction was completed (Rf= 0.47 in ethyl acetate). The solution was diluted with DCM and was treated with ice cold HCL (5M) and then with 45% Na2S205 and water. After drying the organic layer with MgS04, filtering and concentrating in vacuo, compound 16 was purified using flash chromatography. This afforded pure 17 in 57 % yield (329 mg, 0.8 mmol). Rf = 0.39 (Hexane:Ethyl acetate, 4:1 , v:v). [a]D 25 = 63.1 (c = 0.01 , CH3CI3), 1H NMR (600 MHz, CDCI3): 5= 7.25 (JCHCCH2 cHcoMe = 8.4 Hz, d, 2H, CHCCH?, PMB), 6.87 (JcHcoMe CHCCH2 = 8.7 Hz, d, 2H, CHCOMe. PMB), 5.12 (s, 1 H, H1 ), 4.88 (JH3 ,H2 = 5.9 Hz, JH3 ,H4' = 3.9 Hz, dd, 1 H, H3 ), 4.63 (JH2 , H3 = 5.7 Hz, d, 1 H, H2 ), 4.59 (JCHA, CHB = 1 1.6 Hz, ABX, 1 H, CHA, PMB), 4.43 (JCHB CHA = 1 1.6 Hz, ABX, 1 H, CHB, PMB), 4.34 (s, CH2CN), 4.13 (JH4 ,H3 =
3.6 Hz, JH4 ,H5 = 7.1 Hz, dd, 1 H, H4 ), 4.05-3.98 (H5' , H6 ), (JH7 ,H6 = 2.4 Hz, JH7A ,H7B = 9.8 Hz, dd, 1 H, H7A ), 3.82-3.77 (4H, m, CH30, PMB and H7B ), 1.47 (3H, s, CH3COCH2' ,OCH3), 1.31 (3H, s, CH£COCH2' ,OCH3). 13C NMR (150MHz, CDCI3) d= 159.8 (COMe, PMB), 130.0 (CHCCH2 PMB), 129.5 (C PMB), 1 16.1 (CN), 1 14.3 (CHCCH2 PMB), 1 13.1 (C2 ,3 Me02C), 105.7 (C1 ), 85.0 (C2 ), 80.8 (C3 ), 79.4 (C4 ), 72.9 (C7 ),
72.6 (C6 ), 70.5 (C5 ), 69.3 (CH2PMB), 57.2 (CH2CN), 55.6 (OMe, PMB), 26.3 and 24.9 (CH3COCH2 ' ,OCH3 '). HRMS m/z Calcd for C2oH27N08Na [M+Na]+ 432.1634, found 432.16451.
Methoxybenzyl-7-0-(FMOC-aminoethyl)-2,3-0-isopropylidene-D-glycero-a-D-manno heptofuranoside (19).
[00123] Nitrile 17 (196 mg, 479 pmol) was coevaporated with toluene and then dissolved in THF (5 mL). To the mixture was added slowly borane-dimethyl sulfide (182 mI_, 1.9 mmol). After 3 hours at 80°C, the solution was cooled at 0 °C and methanol was slowly added (5 mL). After 16 hours, the solution was concentrated in vacuo and the compound was dissolved in dioxane (1.3 mL) and a NaHC03 (sat. aq.) solution (2.6 mL). FMOCCI (258 mg, 1 mmol) was then added and the reaction was stirred for another 16 hours after which the solution was diluted using water and dichloromethane and the organic layer was washed with water and brine. After drying with MgS04 and concentrating the organic layer in vacuo, the compound was purifier using flash chromatography and afforded pure 19 in 61 % yield over two steps (187 mg, 294 pmol). Rf = 0.45 (Ethyl Acetate:Hexane, 4: 1 , v:v). 1H NMR (600 MHz, CDCI3): d= 7.76 (JHa, Hc = 7.2 Hz, d, 2H, Ha, FMOC), 7.60 (JHb, Hd = 7.7 Hz, d, 2H, Hb, FMOC), 7.39 (JHc, Ha = JHC, Hd = 7.4 Hz, dd (apt), 2H, He, FMOC), 7.31 (JHd, HC = JHd, Hb = 7.4 Hz, dd (apt), 2H, Hd, FMOC), 7.23 (JQHCCH2, CHCOMe = 8.7 Hz, d, 2H, CHCCH?. PMB), 6.86 (JcHcoMe , CHCCH2“ 8.7 Hz, d, 2H, CHCOMe. PMB), 5.25 (NH), 5.12 (s, 1 H, H1 ), 4.89 (JH3 H2 = 5.8 Hz, JH3 ,H4' = 3.6 Hz, dd, 1 H, H3 ), 4.62 ( JH2 , H3 = 5.9 Hz, d, 1 H, H2 ), 4.58 (JCHA, CHB = 1 1.4 Hz, ABX, 1 H, CHA, PMB), 4.42 (JCHB CHA = 1 1.6 Hz, ABX, 1 H, CHB, PMB), 4.39 (m, 2H, ABX, Hf), 4.22 (m, 1 H, He), 4.14 (JH4 ,H3 = 3.6 Hz, JH4 ,H5 = 7.3 Hz, dd, 1 H, H4 ), 4.05 (JH4 .HS = JHS ,H6 = 6.6 Hz, 1 H, H5 ), 3.95 (m, 1 H, H6 ), 3.79 (3H, s, CH30, PMB), 3.61 (m, 2H, H7 ), 3.72 (m, 2H, CH2CH2NH), 3.42 (m, 2H, CH^NH), 1.46 (3H, s, CH3COCH2' ,OCH3), 1.30 (3H, s, CH3COCH2 ' ,OCH3) . 13C NMR (150MHZ, CDCI3) 5= 159.8 (COMe, PMB), 144.3
(CH2CHC), 141.6 (CH2CHCC), 130.0 (CHCCH2 PMB), 129.5 (C PMB), 128.0 (CHc), 127.4 (CHd), 125.4 (CHb), 120.3 (CHa), 1 14.3 (CHCCH2 PMB), 1 13.0 (C2' ,3'Me02C), 105.6 (C1 ), 85.1 (C2 ), 80.8 (C3 ), 79.4 (C4 ), 72.5 (CH2CH2NH), 73.3 (C6 ), 71.0 (C5 ), 70.9 (C7 ), 69.2 (CH2PMB), 67.1 (Cf), 55.6 (OMe, PMB), 47.6 (Ce), 41.1 (CH2NH), 26.3 and 24.8 (CH3COCH2' ,OCH3 ). HRMS m/z Calcd for C34H39NOi4Na [M+Na]+ 658.2628, found 658.2696.
[00124] Compound 19 (60 mg, 94 pmol) was stirred with DCM (5 mL) and water (1 mL) at 0°C. TFA (5 mL) was then added and the reaction stirred for 3 hours. After completion was shown on TLC with (Rf = 0, ethyl acetate), the reaction was concentrated and coevaporated with toluene 6 times. The mixture was dissolved in anhydrous anhydrous pyridine (1 mL) and acetic anhydride (1 mL) and stirred for 16 hours at RT. DCM was added and the organic layer was washed with NaHC03 (sat. aq.) until a neutral pH was reached and then washed with brine. The organic layer was dried over Na2S0 and concentrated in vacuo, the compound was columned using flash chromatography to obtain compound 21 (1 :0.5, a:b) in 81% yield over two steps (53 mg, 77 pmol). 1H NMR (600 MHz, CDCI3): d= 7.76 (JHa, HC = 7.7 Hz, d, 2H, Ha, FMOC), 7.64 (m, 2H, Hb, FMOC), 7.40 (JHc, Ha = JHC, Hd = 7.3 Hz, dd (apt) , 2H, Hc, FMOC), 7.30 (m, 2H, Hd, FMOC), 6.06 (d, Jm a,H2 a = 2.0 Hz, 1 H, HTa), 5.82 (bs, 1 H, H 1 b) , 5.65 (NH), 5.51-5.44 (m, 2H, H4'a and H2b), 5.41 (JH4 p,H3 p = H4 b H2 b = 9.9 Hz, dd (apt), 1 H, H4' b), 5.32 (m, 1 H, H3'a), 5.24 (JH2 a, H1 = 2.2 Hz, JH2 a,H3 a = 3.3 Hz, dd, 1 H, H2'c(), 5.17-5.05 (m, 3H, H6'a, b and H3b), 4.42 (JHfA ,Hg = 7.3 10.4 Hz, 1 H, ABX, HfA), 4.32 (JHfB ,He = 7.1 Hz, JHfB ,HfA =
10.0 Hz, 1 H, ABX, HfB), 4.22 (m, 1 H, He), 4.08 (JH5 a,H4 a = 9-9 Hz, JH5 a,H6 a = 2.6 Hz, dd, H5'a), 3.88-3.73 (m, 1 H, H7Ά, H5b), 3.62 (m, 1 H, H7 B), 3.53 (m, 2H, CFbC^NH), 3.41 (m, 2H, CFbNH), 2.2-1.9 (CH3). 13C NMR (150MHz, CDCIs) d= 171.4, 170.5, 170.4, 170.3, 170.2, 170.1 , 169.8, 168.7, 168.4 (C(0)CH3), 156.9 (OC(O)), 144.4, 144.3 (CH2CHC x 2),
141.6 (CH2CHCC x 2), 128.0 (CHc x 2), 127.4 (CHd x 2), 125.5 (CHb), 120.3 (CHa x 2), 90.7 (CTa), 90.8 (ΰ1 'b), 74.8 (ΰ5' b), 72.4 (C5'a), 71.1-70.7 (ΰ3'b, C6'a, ΰq'b, CH2CH2NH), 69.3 (C3'a), 68.6 (C2'a), 68.5-68.1 (C7' and ΰ2'b), 67.1 (Cf), 66.7 (C4'a),
66.6 (ΰ4'b), 47.7 (He), 41.1 (CH2NH), 21.3-20.8 (CH3). HRMS m/z Calcd for C34H39NOi4Na [M+Na]+ 708.2268, found 708.22804.
[00125] Compound 21 (52.7 mg, 77 pmol) was dissolved into DCM (200 mI_) and to this was added acetic acid (100 mI_) and HBR in acetic acid (500 mI_) under a nitrogen atmosphere. The reaction was stirred for 16 hours at RT after which TLC monitoring showed that the reaction was to completion (Rf = 0.7, diethylether). The mixture was then poured into iced water and extracted with DCM, washed with NaHC03 (sat. aqueous) and water. After this, the reaction mixture was diluted with DCM, washed with NaHC03 (sat. aq.) and water, dried with MgS04 and concentrated in vacuo. The bromide was dissolved in DCM (2 mL) and water was added (2ml_) as well as silver trif late (40 mg, 155 pmol) and silver carbonate (41 mg, 149 pmol). The bromide was hydrolyzed overnight, diluted with DCM and washed with water. The DCM layer was partitioned, dried with MgS04, filtered and concentrated in vacuo. Column chromatography afforded compound 22 in 56% yield (27.5 mg, 43 pmol). HRMS m/z Calcd for C32H37NOi3Na [M+Na]+ 666.2163, found 666.2164.
[00126] Compound 22 (27.5 mg, 43 pmol) was coevaporated using toluene and dried under vacuum overnight. After 16 hours, 22 was dissolved in anhydrous DCM (1 mL) and DMAP (25.4 mg, 210 pmol) was added. Phosphoryl chloride (155 pL, 845 pmol) was also coevaporated with toluene and was dissolved into DCM (1 mL) and using a syringe pump, dropped at a rate of 0.5 mL/h over 2 hours under N2, the reaction was to completion after 5 hours. The reaction was diluted with DCM and washed with TEAB buffer (until a basic pH was reached), water and brine. The organic layer was then dried with Na2S0 , filtered and concentrated under reduced pressure. Flash chromatography using hexane: diethyl ether gave title compound 23b in 28 % yield (10.7 mg, 12 pmol, a:b, 3:2). 1H NMR, (600 MHz, CDCb) 5= 7.76 (JHa, Hc = 7.7 Hz, d, Ha, FMOC), 7.64 (JHb, Hd = 7.3 Hz, d, Hb, FMOC), 7.40-7.27 (m, 10H, He and Hd FMOC and Ph), 7.24-7.15 (m, 4H, Ph), 5.76 (m, NH), 5.65 (JHI ,P = 6.8 Hz, dd, 1 H, HT), 5.46 (m, 1 H, H2 ), 5.37 (JH4 ,H3 = JH4 HS = 9.3 Hz, dd (apt), 1 H, H4 ), 5.19 (m, 1 H, H6 ), 5.05 (JH3 ,H2 = 3.1 Hz, JH3 ,H4 = 8.6 Hz, dd, 1 H, H3 ), 4.42 (JhlfA.B ,Hg = 7.1 HZ, JhlfA ,HfB = 10.9 HZ, ABX, Hf), 4.21 (Jhlg ,HfA = Jhlg ,HfB = 7.1 HZ, ABX, Hg), 3.90 (JH5 ,H6 = 4.8 Hz, JH 5 ,H4 = 9.1 Hz, dd, 1 H, H5 ), 3.69 (JH7A ,H6 = 4.7 Hz, JH7A ,H7B = 10.4 Hz, ABX, H7A ), 3.56 (JH7B ,H6 = 5.5 Hz, JH7B ,H7A = 10.7 Hz, ABX, H7B ), 3.52 (m, ABX, 1 H, CHACH2NH), 3.43 (m, ABX, 1 H, CHBCH2NH), 3.38 (m, 2H, CHgNH), 2.10, 2.09, 2.06, 1 .97 (CH3). 13C NMR (150MHZ, CDCI3) d= 170.4, 170.1 , 170.0 (C(0)CH3), 157.0 (OC(O)), 150.7, 150.3 (CO,Ph x 2), 144.4, 144.3 (CH2CHC x 2), 141.7 (CH2CHCC x 2), 130.3, 130.1 (CHOPh), 128.0 (CHc x 2), 127.4 (CHd x 2), 126.2, 126.1 (CHOPh), 125.6, 125.5 (CHb), 120.6, 120.5 (CHOPh), 120.3 (CHa x 2), 95.2 (2J3I P, CI = 5.0 Hz, C1 ), 74.2 (C5 ), 70.8 (C6 ), 70.6 (CH2CH2NH), 70.3 (C3 ), 68.2 (C7 ), 68.1 (3J3 i P, C2 , = 9.2 Hz, C2 ),
67.1 (Cf), 66.6 (C4 ), 47.7 (He), 41.1 (CH2NH), 21.3, 21.2, 20.9, 20.8 (CH3). Jcr, Hi = 170 Hz. 31P NMR (242 MHz, CDCI3) d -14.0. HRMS m/z Calcd for C H4 NOi6PNa [M+Na]+ 898.2452, found 893.2485.
7-0-(Aminoethyl)-D-glycero^-D-manno-heptopyranose phosphate (JS10a).
[00127] Compound 23b (7.7 mg, 8.8 pmol) was stirred in anhydrous methanol at RT with Pt02 under balloon pressure for 48 hours. When activated, the brown Pt02 turns to black. After filtration over celite and concentration, the compound was dissolved into methanol:water:triethylamine (7:3:1 , v:v:v) at a pH of 1 1 for 4 hours after which it was concentrated and freeze dried over water. Purification on a desalting column (G-10) and exchanging the triethylamine ions for sodium ions gave compound JS10a in 30 % yield (0.9 mg, 2.6 pmol). 1H NMR, (600 MHz, D20) d= 5.05 (JHi 8.7 Hz, d, 1 H, H1 ), 4.13 (m, 1 H, H6 ), 3.99 (JH2 ,H3 = 2.8 Hz, d, 1 H, H2 ), 3.87-3.73 (m, 4H, H7' and CH2CH2NH2), 3.68 (JH3 ,H2 = 3.1 Hz, JH3 ,H4 = 9.7 Hz, dd, 1 H, H3 ), 3.65 (JH4 ,H3 = JH4 ,H5 = 9.8 Hz, dd (apt), 1 H, H4 ) , 3.52 (JH5 ,H6 = 2.9 Hz, JH5 ,H4 = 9.6 Hz, dd, 1 H, H5 ), 3.21 (JCH2,OCH2A = J CH2,OCH2A = 5 Hz, apt, 1 H, Ch NH^. 13C NMR (from HSQC, D20) d= 93.3 (C1 '), 77.2 (C5 ), 72.8 (CH3 ), 71.2 (C2 ), 70.2 (C7 ), 69.2 (C6 ), 67.4 (C4 ), 66.7 (CH2CH2NH), 38.9 (CH2NH2). MS m/z Calcd for C9HI9NOI0P [M-H]- 332.0752, found 332.0758. 7-0-(Aminoethyl)-D-glycero-D-manno-heptopyranose (JS11a).
[00128] Compound 19 (60.5 mg, 95 pmol) was treated with DCM, TFA and water (5:5:1, v:v:v). After 1 hour, the compound was concentrated and purified using reverse phase. The FMOC protected compound was then dissolved into DMF (800 pl_) and piperidin (200 mI_). After an hour, the compound was concentrated in vacuo and few drops of HCI (5 M) were added to the compound and it was desalted on a G-10 column. Pure JS11a was obtained in 51% overall yield (12.4 mg, 49.0 pmol).1H NMR, (600 MHz, D2O) 5= 5.16 (JH1 a, H2 a= 1.7 Hz, d, 1 H, H1 a), 4.87 (JHrp, H2 p= 1.1 Hz, d, 1H, H1'b), 4.22-4.17 (m, 2H, H5'a and H5'b), 3.94 (JH2 p,H3 p = 3.2 Hz, JH2 b,Hi P = 1.0 Hz, d, 1H, H2'b), 3.92
(^H2 a,H3 a= 3.3 Hz, H2 a,H1a = 2.0 Hz, dd, 1H, H2'C(), 3.90 (JH5a,H6 a= 3.0 Hz, JH5a,H4a = 9.6 Hz, dd, 1 H, H5'a), 3.84-3.77 (m, 5H, H3'a, H7A', CH2CH2NH2), 3.75 (
= 9.7 Hz, dd (apt), 1H, H4'a), 3.73-3.67 (H7B', H4'b), 3.64 (JH4 p,H3 p= 3.2 JH4 p,H5 p= 9.4 Hz, dd (apt), 1H, H4'b), 3.46 (JH5p,H6p= 3.0 Hz, JH5p,H4p= 9.5 Hz, dd, 1H, H5'b), 3.24 (JcH2,OCH2A = CH2.0CH2B = 5.4 Hz, apt, 1 H, CH2NH2).13C NMR (600 MHz, D20) 5= 94.0 (C1 a), 93.8 (C1'P), 76.6 (05'b), 73.2 (03'b), 72.6 (C5'a), 71.0 (03'b and C7'a), 70.7 (C7 ' b) , 70.5 (C2'a and C3'a), 70.0 (C6'a), 69.8 (06'b), 67.4 (C4'a), 67.2 (04'b), 66.6 (CH2CH2NH), 38.2 (CH2NH2). HRMS m/z Calcd for C9Hi9N07Na [M+Na]+ 276.1059, found 276.1068. D-glycero-D-manno-heptopyranose (JS12a)
[00129] JS12a was synthesized as described by Brimacombe et al. [9, 10] pH-Rhodo-7-0-(aminoethyl)-D-glycero^-D-manno-heptopyranose phosphate (JS13a)
[00130] JS10a (700 pg) was dissolved in PBS x 1 (100 mI_) and pH-Rhodo activated ester (1 mg in 100 mI_ DMSO) was added to the mixture. After 24 hours, the DMSO the mixture was concentrated in vacuo and JS12a was purified using reverse phase. LRMS and HPLC showed that JS13a was only present at a ratio of 6% in the mixture and was tested according to this. 1H NMR, (500 MHz, D20) 5= 7.39, 7.30, 7.28, 7.09, 7.02, 6.90, 6.88, 6.48, 5.06, 5.04, 4.18, 4.17, 4.16, 4.00, 3.86, 3.73, 3.70, 3.69, 3.68, 3.67, 3.66, 3.64, 3.42, 3.36, 3.35, 3.15, 2.73, 2.68, 2.35, 2.34, 2.32, 2.01 , 1.92, 1.85, 1.83, 1.49, 4.48, 1.41 , 1.38, 1.36, 1.34, 1.30, 1.29, 1.27, 1.26, 1.22, 1.13, 1.12, 1.10, 0.88. LRMS m/z Calcd for
C41H60N5O13P [M+H]+ 861.39, found 861.55.
Biotin-7-0-(aminoethyl)-D-glycero^-D-manno-heptopyranose phosphate (JS14a)
[00131] JS10a (7.2 mg, 21 pmol) was dissolved in water (1 mL) and (+)-Biotin N- hydroxysuccinimide ester (22 mg, 64 pmol in 600 pL DMF) was added to the mixture as well as one drop of Et3N. After 24 hours, the reaction was completed (TLC, rf = 0.32. 5:7:2:2, CHCl3:Me0H:Water:NH40H). Water was added and the water layer was washed three times with ethyl acetate. After the water layer was concentrated and dissolved in brine. This solution was purified on a G-10 column to obtain 2.5 mg (4.5pmol) of JS14a in 21 % yield. 1H NMR, (500 MHz, D20) d= 5.1 1 (JHr,p = 9.0 Hz, d, 1 H, H1 ), 4.62 (m, 1 H, CHCH2S), 4.43 (m, 1 H, CHCHS), 4.17 (m, 1 H, H6 ), 4.00 (m, 1 H, H2 ), 3.79 (m, 1 H, H7Ά), 3.74-3.62 (m, 6H, H7'B,_CH2CH2NH2, H3' , H4 ), 3.49 (m, 1 H, H5 ), 3.41 (m, 1 H, ChbNH^, 3.35 (m, 1 H, CHS), 3.01 (JHA, CHNH = 4.8 Hz, JHA.HB = 13.1 Hz, ABX, 1 H, CHAHBS), 2.79 (JHB, CHNH = 3.6 Hz, JHB,HA = 15.0 Hz, ABX, 1 H, CHAHBS), 2.29 (JCH2C(O), CH2CH2C(0) = 7.3 Hz, 2H, CH2C(0)), 1.8-1.5 (m, 4H, CH2CH2C(0) and ChhCHS), 1.40 (m, 2H, CH2CH2CH2C(0)). 13C NMR (100 MHz, D20) d= 177.1 (C(0)CH2), 95.6 (2J3IP, CI = 4.4 Hz, d, C1 ), 77.4 (C5 ), 72.7 (C3 ), 70.9 (3J3IP, c2 , = 6.8 Hz, d, C2 ), 70.6 (C7 ), 69.9 (C6 ), 69.3 (CH2CH2NH2), 66.9 (C4 ), 62.2 (CHCHS), 60.4 (CHCH2S), 55.4 (CHS), 39.7
(CH2S), 39.0 (CH2NH2), 35.5 (CH2C(0)), 27.9 (CH2CH2CH2C(0)), 27.7 (CH2CHS), 25.1 (CH2CH2C(0)). LRMS mfz Calc’d for CI9H35N30I2PS [M+H]+, 560.1679, found 560.31.
Biotin-PEG4- 7-0-(aminoethyl)-D-glycero^-D-manno-heptopyranose phosphate (JS24a)
[00132] JS10a (4.1 mg, 12 mhioI) was dissolved in water (1 mL) and Biotin-dPEG4-
TFP ester (35 mg, 55 mhioI in 200 mI_ DMF) was added to the mixture. After 24 hours, the reaction was not completed. At this point, 100 mI_ of sat. NaHC03 was added and after 3 hours the reaction was shown to be completed (TLC, rf = 0.32. 5:7:2:2, CHCI3:Me0H:Water:NH40H). The solvents were then evaporated, and the reaction mixture purified on a Biogel P-2 column. 4.8 mg (5.9 pmol) of JS24a was obtained with 50% yield. 1 H NMR, (600 MHz, D20) d= 5.05, 4.62, 4.44, 4.14, 4.00, 3.79, 3.69, 3.48, 3.40, 3.34, 3.00, 2.78, 2.57, 2.30, 1.65, 1.41 LRMS m/z Calc’d for C30H54N4O17PS [M+H]+, 805.29, found 805.19.
Fluorescein-7-0-(aminoethyl)-D-glycero^-D-manno-heptopyranose phosphate (JS25a)
[00133] JS10a (1.5 mg, 4.5 pmol) was dissolved in water (1 mL) and fluorescein N- hydroxysuccinimide ester (5 mg, 8.5 pmol in 200 pL DMF) was added to the mixture. After 24 hours, the reaction did not seem to completion and 100 mI_ of sat. NaHC03 was added. Then after 3 hours, the reaction was completed (TLC, rf = 0.50. 5:7:2:2, CHCl3:MeOH:Water:NH4OH). The DMF and water was then evaporated on a Genevac personal evaporator. The compound was then purified on a biogel P-2 column to obtain 1.6 mg (2.2pmol) of JS25a in 40% yield. 1H NMR, (600 MHz, D20) 5= 8.26, 8.00, 7.62, 7.45, 7.26, 6.90, 6.82, 5.13, 4.17, 4.1 1 , 4.00, 3.77, 3.66, 3.54, 3.47, 3.39, 3.32, 3.28, 2.92, 2.32, 2.24, 1.69, 1.61 , 1.44, 1.34. LRMS m/z Calc’d for C36H40N2O17P [M-H]-, 803.69, found 803.17.
EXAMPLE 2: IN VITRO BIOLOGICAL TESTING
[00134] Immunomodulatory activity was illustrated for compounds JS10a, JS13a, JS14a, JS24a and JS25a according to the data obtained from the experiments highlighted by Figures 9-17.
[00135] The data presented in Figure 9 show that chemically synthesized DD-b-HMR (JS7) elicits an inflammatory response similarto that apparent with DD-b-HBR (JS6) when administered to human colonic epithelial cells (HCT 1 16). The relative absence of activity in cells that lack TIFA (TIFA KO) demonstrates that the activity of both compounds is TIFA-dependent.
[00136] The data presented in Figure 10 shows that chemically synthesized DD-b- HMP (JS7) elicits an inflammatory NF-kB response similar to that apparent with DD-b- HBP (JS6) when administered to human embryonic kidney cells (HEK 293). The relative absence of activity in cells that lack TIFA (TIFA KO) demonstrates that the activity of both compounds is TIFA-dependent.
[00137] The data presented in Figure 11 show that JS-10a elicits a modest inflammatory response whereas JS-13a elicits a strong inflammatory response when administered to human colonic epithelial cells (HCT 1 16). The relative absence of activity in TIFA KO cells demonstrates that the activity of both compounds is TIFA-dependent.
[00138] The data presented in Figure 12 show that JS-10a elicits a modest inflammatory response whereas JS-13a elicits a strong inflammatory response when administered to human embryonic kidney cells (HEK 293). The relative absence of activity in TIFA KO cells demonstrates that the activity of both compounds is TIFA-dependent.
[00139] The data presented in Figure 13 indicate that JS-10a and JS-14a activate similar inflammatory responses when administered to HCT 1 16 colonic epithelial cells. The relative absence of activity in TIFA KO cells indicate that this activity is TIFA- dependent. The Nodi agonist activates a similar response but it is not TIFA-dependent.
[00140] The data presented in Figures 14 indicate that JS-10a and JS-14a activate similar NF-kB responses when administered to HEK 293 human embryonic kidney cells encoding an NF-KB-driven luciferase reporter. The relative absence of activity in TIFA KO cells indicate that this activity is TIFA-dependent. HMP (JS7) is included for comparison of the mono-phosphate with and without a linker at the 7-position.
[00141] The data presented in Figures 15 indicate that JS-10a and JS-14a both stimulate an inflammatory response when higher (150 mM) concentrations are administered to THP-1 human monocytes. HMP (JS7) is included for comparison of the mono-phosphate with and without a linker at the 7-position.
[00142] The data presented in Figure 16 indicate that JS-24a and JS-25a activate similar inflammatory responses when administered to HCT 1 16 colonic epithelial cells. The relative absence of activity in TIFA KO cells indicate that this activity is TIFA- dependent. The Nodi agonist activates a similar response but it is not TIFA-dependent.
[00143] The data presented in Figures 17 indicate that JS-24a and JS-25a activate similar NF-kB responses when administered to HEK 293 human embryonic kidney cells encoding an NF-KB-driven luciferase reporter. The relative absence of activity in TIFA KO cells indicate that this activity is TIFA-dependent. EXAMPLE 3: ADDITIONAL HMP-LINKERS
[00144] Additional compounds (JS 15 to JS19) can be synthesized according to standard techniques such as those described in Li et al., Carbohydrate Research, 2016, 432, 71-75 and Li et al. Organic letters, 2014, 16, 5628-5631.
[00145] For example, other compounds of formula (I) or (la):
can be synthesized, where the R group may be any linkage moiety for conjugating with a molecule, and the resulting conjugate compound is capable of targeting and/or modulating an immune response thereof.
[00146] Particularly, the R group can be selected from the group consisting of: -C(0)OH, -C(0)H, -Co-6alkyl-C(0)-Ci-6 alkyl, -C0-6alkyl-N3, -Ci-6 alkyl, -C0-6alkyl -0-Ci-6 alkyl, -C0-6alkyl-aryl, -Co-6alkyl-0-aryl, -Co-6alkyl-C2-6-allyl groups, and -Co-6alkyl-0-C2-6- allyl, wherein said -C0-6alkyl-C(O)-Ci-6 alkyl, -Ci-6 alkyl, C0-6alkyl-O-Ci-6 alkoxy, -aryl, C0- 6alkyl-0-aryl, C0-6alkyl-C2 b-allyl groups, and Co-6alkyl-0-C2-6-allyl is optionally substituted with a: amino, acyloxy, alkoxy, carboxyl, carbalkoxyl, hydroxy, trifluoromethyl, cyano, nitro, acyl, or a halo group; or R is selected from the group consisting of: CH20P03C(0)-Ci-6 alkyl, CH20P03Ci-6 alkyl, CH20P03-CI-6-0 alkoxy, CH20P03-aryl, CH20P03-C2-6-allyl, wherein said CH20P03C(0)-CI_6 alkyl, CH20P03Ci-6 alkyl, CH20P03-Ci_6-0 alkoxy, CH20P03-aryl, CH20P03-C2-6-allyl group is optionally substituted with a: amino, acyloxy, alkoxy, carboxyl, carbalkoxyl, hydroxy, trifluoromethyl, cyano, nitro, acyl, or a halo group.
[00147] More particularly, the R group can be selected from the group consisting of: - CH2-NH2; -C(0)0H; -C(0)H; - N3; - lower alkyl or -O-lower alkyl groups containing 1-6 saturated or unsaturated carbon atoms; and -aryl, -O-aryl, -O-allyl or -allyl groups having 2-6 carbon atoms; wherein such alkyl, -O-alkyl, aryl, -O-aryl, -allyl or -O-allyl groups may be unsubstituted or may be substituted with : halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl, or amino groups; or with a hydroxy, an amino or a halo group. [00148] Additionally, it is possible to modify HMP via a phosphate residue, wherein R is C0PC>3C(0) Ci-6 alkyl, -COPO3C1-6 alkyl, -COPO3- C1-6-O alkoxy, -COPCb-aryl, - COP03-aryl, -C0P03-C2-6-allyl. More particularly, the compounds of formula (I) as defined herein can be synthesized according to standard techniques, particularly according to US patent 5,856,462.
[00149] The present invention has been described in terms of particular embodiments found or proposed by the present inventor to comprise preferred modes for the practice of the invention. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention. All such modifications are intended to be included within the scope of the appended claims.
[00150] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference in their entirety.
REFERENCES
1. Gaudet, R.G.; Sintsova, A.; Buckwalter, C. M.; Leung, N.; Cochrane, A.; Li, J.; Cox, A.D.; Moffat, J.; Gray-Owen, S.D. Science, 2015, 384, 1251-1255.
2. Gaudet, R.G.; Gray-Owen, S.D. PLOS Pathog. 2016, 72(9): e1005807.
3. Malott, R.J.; O. Keller, B.; Gaudet, R.G.; McCaw, S.E.; Lai, C.C.L.; Dobson- Belaire, W.N.; Hobbs, J. L.; St. Michael, F.; Cox, A.D.; Moraes, T.F.; Gray-Owen, S.D. Proc. Natl. Acad. Sci., 2013, 110, 10234-10239.
4. Milivojevic, M.; Dangeard, A.S.; Kasper, C.A.; Tschon, T.; Emmenlauer, M., Pique, C.; Schnupf, P.; Guignot, J.; Arrieumerlou, C. PLOS Pathog. 2017, 13(2) :e 1006224.
5. Wang, L.; Huang, H.; Nguyen, H. H.; Allen, K. N.; Mariano, P. S.; Dunaway- Mariano, D. Biochemistry, 2010, 49, 1072-1081.
6. Inuke, S.; Aiba, T.; Kawakami, S.; Akiyama, T.; Inoue, J.; Fujimoto, Y. Org. Lett., 2017, DOI: 10.1021/acs.orglett.7b01 158.
7. Rosenfeld, D.A.; Richtmyer, N.K.; Hudson, C.S. J. Am. Chem. Soc., 1951 , 73,
4907-4910. 8. Hulyalkar, R.K.; Jones, J.K.N.; Perry, M.B. Can. J. Chem., 1963, 41, 1490-1492.
9. Brimacombe, J.S.; Kabir, A. K. M. S. Carbohydr. Res., 1986, 152, 329-334.
10. Brimacombe, J.S.; Kabir, A. K. M. S. Carbohydr. Res., 1986, 150, 35-51.
1 1. Guzleck, H.; Graziani, A.; Kosma, P. Carbohydr. Res., 2005, 340, 2808-281 1. 12. Durka, M.; Tikad, A.; Perion, R.; Bosco, M.; Andaloussi, M.; Floquet, S.; Malacain,
E.; Moreau, F.; Oxoby, M.; Gerusz, V.; Vincent, S. P. Chem. Eur. J. 2011 , 77, 1 1305 - 1 1313.
13. Zamyatina, A.; Gronow, S.; Puchberger, M.; Graziani, A.; Hofinger, A.; Kosma, P. Carbohydr. Res., 2003, 338, 2571-2589.
14. Sabesan, S.; Neira, S. Carbohydr. Res. 1992, 223, 169-185.
15. Crich, D.; Dudkin, V. Org. Lett., 2000, 2, 3941-3943.
16. Li,T. ; Tikad, A.; Pan, W.; Vincent, S.P. Org. Lett., 2014, 16, 5628-5631.
17. Gaudet, R.G.; Guo, C. X.; Molinaro, R.; Kottwitz, H.; Rohde, J. R.; Dangeard, A.- S.; Arrieumerlou, C.; Girardin, S.E.; Gray-Owen, S.D. Cell Rep., 2017, 19, 1418-1430. 18. Sauvageau et al., Carbohydr. Res., 2017, 450, 38-43.
19. Adekoya et al. J. Immunol., 2018, doi:10.4049/jimmunol.1801012.
20 Zhou et al. Nature, 2018, 561 , 123-126.

Claims

1. A compound of formula (I):
wherein R is a linkage moiety for conjugating with a functional molecule, and Y is H or an alkali metal.
2. The compound of formula (I) according to claim 1 , wherein Y is Na.
3. The compound of claim 1 or 2, wherein R is selected from the group consisting of:
-C(0)0H, -C(0)H, -Co-6alkyl-C(0)-Ci-6 alkyl, -Co-6alkyl-N3, -Ci-6 alkyl, -Co-6alkyl -0-Ci-6 alkyl, -Co-6alkyl-aryl, -Co-6alkyl-0-aryl, -Co-6alkyl-C2-6-allyl groups, and -Co-6alkyl-0-C2-6- allyl,
wherein said -Co-6alkyl-C(0)-Ci-6 alkyl, -Ci-e alkyl, Co-6alkyl-0-Ci-6 alkoxy, -aryl, Co- 6alkyl-0-aryl, C0-6alkyl-C2 b-allyl groups, and Co-6alkyl-0-C2-6-allyl is optionally substituted with a: amino, acyloxy, alkoxy, carboxyl, carbalkoxyl, hydroxy, trifluoromethyl, cyano, nitro, acyl, or a halo group;
or R is selected from the group consisting of: -Ci-6 alkyl-0P03C(0)-Ci-6 alkyl, -Ci-6 alkyl- 0P03Ci-6 alkyl, -Ci-6 alkyl-OP03-Ci-6-0-alkoxy, -Ci-6 alkyl-OP03-aryl, -Ci-6 alkyl-0P03-C2- 6-allyl,
wherein said -Ci-6 alkyl-0P03C(0)-Ci-6 alkyl, -Ci-6 alkyl-0P03Ci-6 alkyl, -Ci-6 alkyl- OP03-Ci-6-0-alkoxy, -Ci-6 alkyl-OP03-aryl, -Ci-6 alkyl-0P03-C2-6-allyl group is optionally substituted with a: amino, acyloxy, alkoxy, carboxyl, carbalkoxyl, hydroxy, trifluoromethyl, cyano, nitro, acyl, or a halo group;
or a salt thereof.
4. The compound of claim 1 , 2 or 3, wherein R is selected from the group consisting of:
-C(0)0H, -C(0)H, -C(0)-Ci-s alkyl, -N3, -Ci-6 alkyl, -0-Ci-6 alkyl, -aryl, -O-aryl, -C2-6-allyl groups, and -0-C2 e-allyl; wherein said -C(0)-Ci-6 alkyl, -Ci-6 alkyl, -0-Ci-6 alkoxy, -aryl, -O-aryl, -C2-6-allyl groups, and -0-C2-6-allyl is optionally substituted with : amino, acyloxy, alkoxy, carboxyl, carbalkoxyl, hydroxy, trifluoromethyl, cyano, nitro, acyl, or a halo group; or R is selected from the group consisting of: -CH20P03C(0)-CI-6 alkyl, -CH2OPO3C1 -6 alkyl, -CH2OPO3-C1-6-O alkoxy, -CH20P03-aryl, -CH20P03-C2-6-allyl,
wherein said -CH20P03C(0)-CI-6 alkyl, -CH2OPO3C1-6 alkyl, -CH2OPO3-C1-6-O alkoxy, -CH20P03-aryl, -CH20P03-C2-6-allyl group is optionally substituted with a: amino, acyloxy, alkoxy, carboxyl, carbalkoxyl, hydroxy, trifluoromethyl, cyano, nitro, acyl, or a halo group;
or a salt thereof.
5. The compound of any one of claims 2 to 4, wherein R is selected from the group consisting of: -Ci-ealkyl, -C(0)0H, -C(0)H, -C(0)-Ci-e alkyl, -Co-6alkyl-0-Ci-6 alkyl, -Co- 6alkyl-C2-6-allyl groups, and -0-C2-6-allyl;
wherein each of said -Ci-6alkyl, -C(0)-Ci-6 alkyl, -C0-6alkyl-O-Ci-6 alkyl, _-C0-6alkyl- C2-6-allyl, and -Co-6alkyl-0-C2-6-allyl group is optionally substituted with a : amino, alkoxy, hydroxy, carboxy or nitro group;
or R is selected from the group consisting of: -CH20PC>3C(0)-CI-6 alkyl, -CH2OPO3C1 -6 alkyl, -CH2OP03-Ci-6-0-alkoxy, -CH20P03-aryl, -CH20P03-C2-6-allyl,
wherein each of said -CH20P03C(0)-CI-6 alkyl, -CH2OPO3C1 -6 alkyl, -CH2OPO3- Ci-6-O-alkoxy, -CH20P03-aryl, -CH20P03-C2-6-allyl group is optionally substituted with a: amino, alkoxy, hydroxy, or nitro group;
or a salt thereof.
6. The compound of claim 5, wherein R is selected from the group consisting of: Ci-6alkyl-NH2; -C0-6 alkyl-C-O-Co-e alkyl-NH2, -C(0)0H, -C(0)H, -C0-6 alkyl-C(O)C0-6 alkyl- NH2, Co-ealkyl-Ns.
7. The compound of claim 6, wherein R is selected from the group consisting of:CH2- NH2; -C(0)0H, -C(0)H, -C(0)NH2 and - N3.
8. The compound of claim 7, selected from the group consisting of:
JS17
JS18 ; and JS19
9. A compound comprising a functional molecule conjugated to a D-glycero-D- manno-heptopyranose I b-phosphate (heptopyranose-1 -monophosphate; HMP) moiety.
10. The compound of claim 9, wherein the functional molecule is conjugated at position-7 of the HMP.
11. The compound of claim 9, wherein the functional molecule is conjugated at position-2 of the HMP.
12. The compound of claim 9, being
wherein X is a conjugated functional molecule.
13. The compound of claim 10, wherein the conjugated functional molecule is selected from the group consisting of a: protein, polypeptide, antibody, lipid, fatty acid, carbohydrate, drug, transfer factor and marker.
14. The compound of claim 13, wherein the marker is selected from the group consisting of: a label and a tag.
15. The compound of claim 14, wherein the label or tag is selected from the group consisting of: biotin, rhodamine, fluorescein, CF tags, EvoBlue, rhodopsin, GFP, fluorescent red, fluorescent orange, biotin, atto labels, carbohydrate labels, CF dyes, mega labels, Phycobili Proteins, tracy labels and metal complexes, or derivatives thereof.
16. The compound of claim 15, being selected from the group consisting of:
17. The compound of claim 12, wherein the conjugated functional molecule is an antigen.
18. The compound of claim 17, wherein the antigen is a cell differentiation binding partner or an immunogen.
19. The compound of claim 18, wherein the antigen is derived from immune cells.
20. The compound of claim 17, wherein the antigen is an immunogen derived from bacteria, virus or parasites.
21. The compound of claim 20, wherein the immunogen is from Gram-negative bacteria.
22. The compound of claim 21 , wherein the Gram-negative bacteria are selected from the group consisting of: Neisseria, Escherichia, Klebsiella, Salmonella, Shigella, Vibrio, Helicobacter, Pseudomonas, Burkholderia, Haemophilus, Moraxella, Bordetella, Francisella, Pasteurella, Borrelia, Campylobacter, Yersinia, Rickettsia, Treponema, Chlamydia and Brucella.
23. The compound of claim 18, wherein the immunogen is from Gram-positive bacteria.
24. The compound of claim 22, wherein the Gram-positive bacteria are selected from the group consisting of: Staphylococcus, Streptococcus, Listeria, Corynebacterium, Enterococcus, Clostridium and Mycobacterium.
25. The compound of claim 18, wherein the immunogen is from a virus selected from the group of viruses selected from the group consisting of: Adenoviridae, Herpesviridae, Papillomviridae, Poxviridae, Hepadnaviridae, Parvoviridae, Astroviridae, Caliciviridae, Picornaviridae, Coronaviridae, Flaviviridae, Togaviridae, Flepeviridae, Retroviridae, Orthomyxoviridae, Arenaviridae, Bunyaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, Reoviridae and Human Immunodeficiency virus (HIV).
26. The compound of claim 18, wherein the immunogen is from a parasite selected from the group of: Leishmania, Plasmodium, Toxoplasma, Trypanosoma and Schistosoma.
27. The compound of claim 17, wherein the antigen is a cancer-specific antigen.
28. The compound of claim 17, wherein the conjugated functional molecule is a cancer-specific antigen binding partner.
29. The compound of claim 13, wherein the functional molecule conjugated to the HMP-linker is an anti-cancer or an anti-viral drug.
30. The compound of claim 29, wherein the anti-cancer drug is selected from the group consisting of: Abiraterone, Alemtuzumab, Anastrozole, Aprepitant, Arsenic trioxide, Atezolizumab, Azacitidine, Bevacizumab, Bleomycin, Bortezomib, Cabazitaxel, Capecitabine, Carboplatin, Cetuximab, Chemotherapy drug combinations, Cisplatin, Crizotinib, Cyclophosphamide, Cytarabine, Denosumab, Docetaxel, Doxorubicin, Eribulin, Erlotinib, Etoposide, Everolimus, Exemestane, Filgrastim, Fluorouracil, Fulvestrant, Gemcitabine, HPV Vaccine, Imatinib, Imiquimod, Ipilimumab, Ixabepilone, Lapatinib, Lenalidomide, Letrozole, Leuprolide, Mesna, Methotrexate, Nivolumab, Oxaliplatin, Paclitaxel, Palonosetron, Pembrolizumab, Pemetrexed, Prednisone, Radium-223, Rituximab, Sipuleucel-T, Sorafenib, Sunitinib, Talc Intrapleural, Tamoxifen, Temozolomide, Temsirolimus, Thalidomide, Trastuzumab, Vinorelbine, and Zoledronic acid.
31. The compound of claim 29, wherein the anti-viral drug is an anti-HIV drug.
32. The compound of claim 31 , wherein the anti-HIV drug is selected from the group consisting of: Nucleoside/Nucleotide reverse transcriptase inhibitors (NRTI), Nonnucleoside reverse transcriptase inhibitors (NNRTIs), Protease inhibitors (PI), Entry inhibitors, Chemokine co-receptor antagonists (CCR5 antagonists), Multiclass combination drug and Cytochrome P4503A (CYP3A) inhibitors.
33. The compound of claim 32, wherein the Nucleoside/Nucleotide reverse transcriptase inhibitors (NRTI) is selected from the group consisting of: abacavir (ziagen), efavirenz/emtriacitabine/tenofovir disoproxil fumarate (atripla), lamivudine/zidovudine (combivir), emtriacitabine/rilpivirine/tenofovir disoproxil fumarate (complera), emtricitabine (emtriva), lamivudine (Epivir), abacavir/lamivudine (Epzicom), zidovudine (Retrovir), abacavir/lamivudine/zidovudine (Trizivir), emtricitabine/tenofovire disoproxil fumarate (Truvada), didanosine (Videx), didanosine extended release (Videx EC), tenofovir disoproxil fumarate (Viread) and stavudine (Zerit).
34. The compound of claim 32, wherein the Multiclass combination drug is selected from the group consisting of: efavirenz/emtricitabine/tenofovir disoproxil fumarate (Atripla), emtricitabine/rilpivirine/tenofovir disoproxil fumarate (Complera), elvitegravir/cobicistat/emtricitabine/tenofovir disoproxil fumarate (Stribild), and abacavir /dolutegravir/lamivudine (Triumeq).
35. The compound of claim 32, wherein the Non-nucleoside reverse transcriptase inhibitors (NNRTIs) drug is selected from the group consisting of: rilpivirine (Edurant), etravirine (Intelence), delavirdine mesylate (Rescriptor), efavirenz (Sustiva), nevirapine (Viramune) and nevirapine extended-release (Viramune XR).
36. The compound of claim 32, wherein the protease inhibitors (PI) drug is selected from the group consisting of: tipranavir (Aptivus), indinavir (Crixivan), atazanavir/cobicistat (Evotaz), saquinavir (Invirase), lopinavir/ritonavir (Kaletra), fosamprenavir (Lexiva), ritonavir (Norvir), darunavir/cobicistat (Prezcobix), darunavir (Prezista), atazanavir (Reyataz) and nelfinavir (Viracept).
37. The compound of claim 32, wherein the entry inhibitor drug is selected from the group consisting of: enfuvirtide (Fuzeon).
38. The compound of claim 32, wherein the integrase inhibitor drug is selected from the group consisting of: raltegravir (Isentress), dolutegravir (Tivicay) and elvitegravir (Vitekta).
39. The compound of claim 32, wherein the chemokine co-receptor (CCR5) antagonist is selected from the group consisting of: maraviroc (Selzentry).
40. The compound of claim 32, wherein the anti-viral drug is selected from the group consisting of: simeprevir (Olysio), boceprevir (Victrelis), and ombitasvir/paritaprevir/ritonavir/dasabuvir (Viekira Pak).
41. A method to prepare a therapeutic conjugate with modulated immunomodulatory activity comprising the step of: conjugating a molecule with a compound of formula (la):
wherein R is as defined in any one of claims 2 to 7, or a salt thereof.
42. A compound of formula (II):
wherein X is a functional molecule and Y is H or an alkali metal; a salt thereof, for use in targeting or modulating an immune response in a subject,
43. The conjugate compound of formula (II) for use according to claim 42, wherein the modulating the immune response in the subject is used for preventing, treating, ameliorating, or inhibiting an injury, disease, disorder or condition wherein the modulation of the immune response is beneficial.
44. The conjugate for use of claim 43, wherein the immune response of the subject is enhanced.
45. The conjugate for use of claim 44, wherein the use is carried out as a vaccine for the treatment or prevention of disease.
46. The conjugate for use of claim 44, wherein the use is for the treatment or prevention of cancer or viral infections.
47. The conjugate for use of claim 46, wherein the viral infection is caused by HIV.
48. The conjugate for use of claim 42, wherein the immune response of the subject is decreased.
49. The conjugate for use of claim 48, wherein the decreased immune response decreases inflammation in subject.
50. The conjugate for use of claim 48, for inhibiting an allergic reaction in a subject.
51. The conjugate for use of claim any one of claims 42 to 50, wherein the subject is a human.
52. Use of an effective amount of a compound of formula (II):
wherein X is a conjugated molecule and Y is H or an alkali metal; or a salt thereof, for targeting or modulating an immune response in a subject.
53. The use of claim 52, wherein modulating comprises preventing, treating, ameliorating, or inhibiting an injury, disease, disorder or condition wherein modulation of the immune response is beneficial.
54. Use of a compound of formula (II):
wherein X is a conjugated molecule and Y is OH or an alkali metal; or a salt thereof, for the manufacture of a medicament for modulating an immune response in a subject.
55. A method for modulating an immune response in a subject, comprising administering an effective amount of the compound of formula (II) as defined in claim 54.
56. The use of claim 54, wherein the immune response of the subject is enhanced.
57. The use of claim 54, wherein the compound is a vaccine for the treatment or prevention of disease.
58. The use of claim 54, wherein the immune response of the subject is decreased.
59. The use of claim 58, wherein the decreased immune response decreases inflammation in subject.
60. The use of claim 58, for inhibiting an allergic reaction in a subject.
61. The use of claim 54, wherein the subject is a human.
62. A method for the treatment of HIV comprising the steps of:
• administering an HMP-derivative conjugated to an anti-HIV molecule, wherein the HMP moiety stimulates HIV latently-infected cells to start producing virus, and the anti-HIV molecule kills the produced virus;
whereby the HMP-anti-HIV conjugate eradicates the latent viral pool and treats the HIV infection.
63. A method for the treatment of HIV comprising the steps of:
• administering an HMP-derivative conjugated to an HIV-reservoir cell-specific targeting molecule to a subject, wherein the conjugate-HMP moiety stimulates HIV latently-infected cells to start producing virus, and
• administering an effective dose of an anti-HIV drug to the subject to kill the produced virus;
whereby the HMP-anti-HIV conjugate eradicates the latent viral pool and treats the HIV infection.
64. The method of claim 62 or 63, wherein said anti-HIV molecule is selected from the group consisting of: Nucleoside/Nucleotide reverse transcriptase inhibitors (NRTI), Non-nucleoside reverse transcriptase inhibitors (NNRTIs), Protease inhibitors (PI), Entry inhibitors, Chemokine co-receptor antagonists (CCR5 antagonists), Multiclass combination drug and Cytochrome P4503A (CYP3A) inhibitors.
65. A method for synthesizing a compound of formula (II) comprising the steps of:
a) protecting hydroxy group of compound #7 to yield compound #16;
b) hydroxylating double bond of compound #16 to yield compound #17;
c) reducing nitrile of compound #17 to an amine of compound #19 and protecting compound #19 with a P2 protecting group;
d) cleaving acetonide and P1 protecting group and acetylating to yield acetylated compound #21 ;
e) deprotecting anomeric acetyl of compound #21 to yield compound #22;
f) phosphorylating compound #22 to yield compound #23;
g) deprotecting compound #23 to form JS10a; and
j) conjugating JS10a to an activated ester of a molecule X to yield conjugate of formula (II).
66. The compound of claim 27, wherein the cancer-specific antigen is selected from the group consisting of:
CEA
Immature laminin receptor NY-ESO-l/LAGE-1
TAG-72 PRAME
HPV E6, E7 SSX-2
BING-4
Calcium-activated chloride channel 2
Cyclin-Bi
9D7
Ep-CAM
BAGE family
EphA3
Her2/neu CAGE family
Telomerase GAGE family
Mesothelin MAGE family
SAGE family
SAP-1
XAGE family
Survivin
BRCA1/2
CDK4
Melan-A/MART-1 CML66
Gp l00/pmell7 Fibronectin
Tyrosinase MART-2
TRP-1/-2 p53
P.polypeptide Ras
MC1R TGF-pRII
Prostate -pecific antigen MUC 1
p-catenin
Ig, TCR
BRCA = breast cancer antigen; CDK4 = cyclin-dependent kinase-4; CEA = carcino-embryonic antigen; CML66 = chronic myelogenous leukemia (antigen) 66; CT= cancer testis; HPV = human papilloma virus; Ep-CAM = epithelial cell adhesion molecule; Ig = immunoglobulin; MART-1/-2 = melanoma antigen recognized by T cells- 1/-2; MC 1R = melanocortin-1 -receptor; SAP-1 = stomach cancer- associated protein tyrosine phosphatase- 1 ; TAG-72 = tumor antigen-72; TCR = T cell receptor; TGF-PRII = transforming growth factor-b receptor II; TRP = tyrosinase-related protein.
EP18896861.4A 2017-12-29 2018-12-21 D-GLYCERO-B-D-HEPTOSE-1-PHOSPHATE (HMP) CONJUGATES AND USE FOR TARGETED IMMUNODULATION Pending EP3732184A4 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201762611640P 2017-12-29 2017-12-29
PCT/CA2018/051652 WO2019126873A1 (en) 2017-12-29 2018-12-21 D-glycero-b-d-heptose 1-phosphate (hmp) conjugates and use for targeted immune modulation

Publications (2)

Publication Number Publication Date
EP3732184A1 true EP3732184A1 (en) 2020-11-04
EP3732184A4 EP3732184A4 (en) 2021-11-24

Family

ID=67062834

Family Applications (1)

Application Number Title Priority Date Filing Date
EP18896861.4A Pending EP3732184A4 (en) 2017-12-29 2018-12-21 D-GLYCERO-B-D-HEPTOSE-1-PHOSPHATE (HMP) CONJUGATES AND USE FOR TARGETED IMMUNODULATION

Country Status (4)

Country Link
US (1) US11795190B2 (en)
EP (1) EP3732184A4 (en)
CA (1) CA3086690A1 (en)
WO (1) WO2019126873A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20230110501A (en) * 2020-10-01 2023-07-24 미시시피 주립대학 Drugs Conjugated with Hexose Phosphates and Methods of Making and Using The Same

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4425098A1 (en) 1994-07-15 1996-01-18 Forsch Borstel Inst Fuer Exper 7-0-carbamoyl heptose derivatives, process for their preparation and their use and screening process for their determination
CA2963724C (en) * 2014-10-10 2021-08-24 The Governing Council Of The University Of Toronto Methods of modulating immune system responses
CA3062714A1 (en) * 2017-05-11 2018-11-15 National Research Council Of Canada Use of phosphorylated heptose compounds
US11707519B2 (en) * 2017-05-11 2023-07-25 University College Dublin, National University Of Ireland Phosphorylated heptose compounds: process for their preparation and use

Also Published As

Publication number Publication date
WO2019126873A1 (en) 2019-07-04
US20200331949A1 (en) 2020-10-22
US11795190B2 (en) 2023-10-24
CA3086690A1 (en) 2019-07-04
EP3732184A4 (en) 2021-11-24

Similar Documents

Publication Publication Date Title
ES2754269T3 (en) Compositions and Activation Methods of "Interferon Gene Stimulator-dependent" Signaling
CN105008381B (en) Compositions comprising cyclic purine dinucleotides of defined stereochemistry and methods of making and using the same
CA3078267C (en) Compositions and methods of modulating the immune response by activating alpha protein kinase 1
CN109451740B (en) Cyclic dinucleotides for the treatment of disorders associated with STING activity, such as cancer
TW201639866A (en) Compositions and methods for activating "stimulator of interferon gene"-dependent signalling
CN109843302A (en) Compounds, compositions and methods for treating diseases
BRPI0617687A2 (en) compound, method for protecting a mammal against, or treating, a virus, microbial infection, parasite, an autoimmune disease, cancer, allergy or asthma, and, pharmaceutical composition
US11795190B2 (en) D-glycero-B-D-heptose 1-phosphate (HMP) conjugates and use for targeted immune modulation
JP7296459B2 (en) Stable vaccine against Clostridium difficile
HK1259906A1 (en) Compositions comprising cyclic purine dinucleotides having defined stereochemistries and methods for their preparation and use
OA21080A (en) Adjuvant with TLR4 agonist activity.
Calabrese et al. Modulation of CD14 and TLR4⋅ MD-2 Activities by a Synthetic Lipid A Mimetic
HK1212707B (en) Compositions comprising cyclic purine dinucleotides having defined stereochemistries and methods for their preparation and use
HK1219024B (en) Compositions and methods for activating "stimulator of interferon gene"-dependent signalling
HK1222512B (en) Compositions and methods for activating "stimulator of interferon gene"-dependent signalling

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20200724

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
A4 Supplementary search report drawn up and despatched

Effective date: 20211026

RIC1 Information provided on ipc code assigned before grant

Ipc: A61P 37/04 20060101ALI20211020BHEP

Ipc: A61P 37/02 20060101ALI20211020BHEP

Ipc: A61P 35/00 20060101ALI20211020BHEP

Ipc: A61P 31/18 20060101ALI20211020BHEP

Ipc: A61K 47/54 20170101ALI20211020BHEP

Ipc: A61K 39/39 20060101ALI20211020BHEP

Ipc: C07H 1/02 20060101ALI20211020BHEP

Ipc: C07H 11/04 20060101AFI20211020BHEP